Multifunctional small molecules

ABSTRACT

The present invention relates to dendrimer synthesis. Specifically, the present invention relates to triazine scaffolds capable of click chemistry for one-step synthesis of functionalized dendrimers, and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. 371 national phase entry of InternationalPatent Application No. PCT/US2010/050893, international filing date Sep.30, 2010 which claims priority to U.S. Provisional Patent ApplicationNo. 61/256,699, filed Oct. 30, 2009, the contents of which are hereinincorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under contract numberR01 CA119409 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to dendrimer synthesis. Specifically, thepresent invention relates to triazine compositions (e.g., scaffolds)capable of click chemistry for use in one-step synthesis offunctionalized dendrimers, and methods of use of the same.

BACKGROUND OF THE INVENTION

Cancer remains the number two cause of mortality in the United States,resulting in over 500,000 deaths per year. Despite advances in detectionand treatment, cancer mortality remains high. New compositions andmethods for the imaging and treatment (e.g., therapeutic) of cancer mayhelp to reduce the rate of mortality associated with cancer.

Severe, chronic pain is observed on a variety of subjects. For example,there exist large numbers of individuals with severe pain associatedwith arthritis, autoimmune disease, injury, cancer, and a host of otherconditions.

There exists a need for compositions, methods and systems for deliveringagents (e.g., diagnostic and/or therapeutic (e.g., cancer therapeutics,pain relief agents) to subjects that provide effective therapy (e.g.,disease treatment, symptom relief, etc.) with reduced or eliminated sideeffects, even when administered in high doses. Functionalizeddendrimers, such as PAMAM dendrimers conjugated with functional ligandsrelevant to cancer therapy and/or pain alleviation, have been developedfor such purposes. However, multi-step conjugation strategies used toattach different functional groups to the surfaces of nanoparticles(e.g., dendrimers, PAMAM dendrimer branches) introduce higherpolydispersity and require multiple processing steps, therebycomplicating synthesis. In addition, increased polydispersity offunctionalized dendrimer products can negatively affect properties suchas therapeutic potency, pharmacokinetics, or effectiveness formultivalent targeting.

Improved methods of synthesis of dendrimers resulting in decreasedpolydispersity are needed. In particular, compositions and methods thatfacilitate one-step “click” chemistry for use in synthesis offunctionalized dendrimers are needed.

SUMMARY OF THE INVENTION

The present invention relates to dendrimer synthesis. Specifically, thepresent invention relates to triazine compositions (e.g., scaffolds)capable of click chemistry for use in one-step synthesis offunctionalized dendrimers and methods of synthesizing and using thesame.

Functionalized dendrimers, such as PAMAM dendrimers conjugated withfunctional ligands (e.g., therapeutic agents, triggering agents, imagingagents, targeting agents) have many therapeutic and diagnosticapplications. However, multi-step conjugation strategies used to attachdifferent functional ligands to the surfaces of nanoparticles (e.g.,dendrimers, PAMAM dendrimer branches) introduce higher polydispersityand require multiple processing steps, thereby complicating synthesisand negatively affecting properties such as therapeutic potency,pharmacokinetics, or effectiveness for multivalent targeting.

For example, multifunctional cancer therapeutics have been developedbased on Generation 5 PAMAM dendrimers (G5) (see, e.g., Majoros, I. J.;et al., J Med Chem 2005, 48, 5892; Thomas, T. P.; et al., J Med Chem2005, 48, 3729; Kukowska-Latallo, J. F.; et al., Cancer Res 2005, 65,5317; Zhang, Y. H.; et al., Bioconjugate Chem 2010, 21, 489; each hereinincorporated by reference in their entireties). The targeting moleculefolic acid (FA) and the chemotherapeutic drug methotrexate (MTX) wereconjugated sequentially through amide- and ester-linkages, respectively(see, e.g., Majoros, I. J.; et al., J Med Chem 2005, 48, 5892; hereinincorporated by reference in its entirety). Although this device wasshown to bind and selectively kill KB tumor cells that over-expressfolate receptor (FR) in vitro and in vivo (see, e.g., Thomas, T. P.; etal., J Med Chem 2005, 48, 3729; Kukowska-Latallo, J. F.; et al., CancerRes 2005, 65, 5317; each herein incorporated by reference in itsentireties), the multi-step conjugation strategy that was employedresulted in a mixture of dendrimer populations having varyingdistributions of the targeting ligands (FA and MTX). The resultingpolydispersity was more severe and problematic during large-scalesynthesis of the conjugate, which hindered attempts to move intoclinical trials. This was because, for example, the polydisperisty ofthe functionalized dendrimer-based therapeutics likely negativelyaffected properties such as therapeutic potency, pharmacokinetics, andeffectiveness for multivalent targeting (see, e.g., Gillies, E. R.; etal., Drug Discov Today 2005, 10, 35; herein incorporated by referenceits entirety). Moreover, multi-step conjugations make batch-to-batchreproducibility extremely challenging, as the characterization requiredafter each step becomes progressively more difficult (see, e.g., Mullen,D. G.; et al., Bioconjugate Chem 2008, 19, 1748; Mullen, D. G.; Acs Nano2010, 4, 657; each herein incorporated by reference in theirentireties). The present invention overcomes such issues and providesimproved methods for the synthesis of well-defined multifunctionaldendrimers.

It has been previously shown that modifications to the side chains ofthe glutamic acid moiety on both FA and MTX can be made without loss ofactivity (see, e.g., Rosowsky, A.; et al., J Med Chem 1981, 24, 1450;Wang, S.; et al., Bioconjugate Chem 1996, 7, 56; each incorporated byreference in their entireties). The simplest approach for coupling FA orMTX to a PAMAM dendrimer is through non-selective activation of thecarboxyl groups of the glutamic acid moiety by carbodiimide (see, e.g.,Majoros, I. J.; et al., J Med Chem 2005, 48, 5892; herein incorporatedby reference in its entirety). Unfortunately, this method leads to theformation of two structural isomers in which FA and MTX are linkedthrough either the α- or γ-carboxyl group. It has been reported that FAlinked via γ-carboxyl group retains a stronger affinity toward itsreceptor (see, e.g., Wang, S.; et al., Bioconjugate Chem 1996, 7, 56;herein incorporated by reference in its entirety). It also has beenshown that a free α-carboxyl group is essential for retaining MTX'sbinding to target enzymes such as dihydrofolate reductase (DHFR),whereas chemical modifications of the γ-carboxyl group are much bettertolerated (see, e.g., Wells, X. E.; et al., Drug Develop Res 1999, 46,302; herein incorporated by reference in its entirety). In addition, theavailability of multiple amino functional groups on the dendrimersurface allows for the nonspecific reaction with the α- and γ-carboxylgroups of MTX and FA, leading to the formation of inactive conjugateswhere both carboxyl groups have been coupled. Furthermore, dendrimerdimers can form with MTX and/or FA serving as a covalent bridge.Therefore, precise control of these coupling reactions is necessary topreserve the therapeutic activity of the conjugate.

The present invention utilizes, for example, triazine scaffolds (e.g.,trivalent reagent 2,4,6-trichloro-1,3,5-triazines as a core scaffold) togive access to multifunctional architectures, thereby enabling replacingseveral chemical entities with a single molecule. Indeed, experimentsconducted during the course of developing some embodiments of thepresent invention determined that multivalent triazine compounds (e.g.,triazine scaffolds) find use in one-step (e.g., click chemistry)synthesis of functionalized dendrimers. Moreover, experiments conductedduring the course of developing some embodiments of the presentinvention demonstrated that such derivatives (e.g., triazine derivativesincorporating the targeting moiety FA and therapeutic drug MTXconjugated to PAMAM dendrimers through “click chemistry”) demonstratedan in vitro dose-dependent cytotoxicity with higher cytotoxic potentialvs. a similar conjugate synthesized by a multi-step approach (see, e.g.,FIGS. 9 and 10 and Example III).

The present invention is not limited to particular triazine compounds(e.g., triazine scaffolds). In some embodiments, the triazine scaffoldscomprise a ring structure (e.g., comprising three nitrogen atoms). Thetriazine scaffolds are not limited to a particular type or kind oftriazine ring structure. In some embodiments, the ring structure issix-membered (e.g., the molecular formula comprises C₃H₃N₃). In someembodiments, the ring is a conjugated system. For example, triazinemoieties with six-membered rings may have nitrogen atoms at any possibleplacement so long as three nitrogen atoms occur in the ring (e.g.,1,2,3-triazine; 1,2,4-triazine, 1,3,5-triazine, 1,2,5-triazine,1,2,6-triazine, etc.). Examples of triazine scaffolds include, but arenot limited to,

(e.g., 2,4,6-trichloro-1,3,5-triazine),

The triazine scaffolds are not limited to a particular manner ofsynthesis. In some embodiments, the triazine scaffolds are synthesizedfrom cyanuric chloride by consecutive aromatic nucleophilic substitutionreactions (e.g., as described in Example 2).

In experiments conducted during the development of embodiments for thepresent invention, trivalent triazine small molecules were used as acore scaffold on which two sites were used for binding (e.g.,conjugation) to functional ligands and a third site was used forconjugation to an azide linker. It was determined that the linker itselfcan be functionalized (e.g., 3-azido-coumarine finds use as an imagingagent). The elaborated triazine scaffold was then used for one-step“click chemistry” reaction with an alkyne-modified PAMAM dendrimer,resulting in a functionalized dendrimer (e.g., nanodevice). It wasdetermined that the azide-containing linker (e.g., 3-azido-coumarine) isfluorescence-silent prior to the click chemistry reaction, andfluoresces after conjugation to the alkyne-modified PAMAM dendrimer,thereby providing a means to monitor dendrimer modification. Moreover,one-step reaction of dendrimer (e.g., modified dendrimers, modifieddendrimer terminal branches) with a multivalent triazine scaffoldresulted in decreased polydispersity of the final functionalizednanodevice product.

The present invention is not limited to utilizing a particular type orform of dendrimer. Indeed, examples of dendrimers finding use in thepresent invention include, but are not limited to, PAMAM dendrimer, aBaker-Huang PAMAM dendrimer (see, e.g., U.S. Provisional PatentApplication No. 61/251,244, herein incorporated by reference in itsentirety), a polypropylamine (POPAM) dendrimer, and a PAMAM-POPAMdendrimer. The type of dendrimer used is not limited by the generationnumber of the dendrimer. Dendrimer molecules may be generation 0,generation 1, generation 2, generation 3, generation 4, generation 5,generation 6, generation 7, or higher than generation 7. In someembodiments, half-generation dendrimers may be used. In certainembodiments, a generation 5 amine-terminated PAMAM dendrimer is used. Incertain embodiments, a generation 5 alkyne-terminated PAMAM dendrimer isused. In some embodiments, the dendrimer is at least partiallyacetylated.

Dendrimers are not limited by their method of synthesis. The dendrimermay be synthesized by divergent synthesis methods or convergentsynthesis methods. In certain embodiments of the present invention,dendrimer molecules may be modified. Modifications may include but arenot limited to the addition of amine-blocking groups (e.g., acetylgroups), ligands, functional groups, conjugates, and/or linkers notoriginally present on the dendrimer. Modification may be partial orcomplete. In some embodiments, all of the termini of the dendrimermolecules are modified. In some embodiments, not all of the dendrimermolecules are modified. In preferred embodiments, methods and systems ofthe present invention permit identification and isolation ofsubpopulations of dendrimers with known numbers of ligand attachments(e.g., conjugations) per dendrimer molecule, thereby yielding samples orsubpopulations of dendrimer compositions with high structuraluniformity.

The present invention is not limited to particular ligand types (e.g.,functional groups) (e.g., for conjugation with dendrimers and/or withtriazine scaffolds). Examples of ligand types (e.g., functional groups)include but are not limited to therapeutic agents, targeting agents,trigger agents, and imaging agents. In some embodiments, the ligand isan alkyne ligand that includes an alkyne group. In some embodiments, theligand is an azide ligand that includes an azido group. In someembodiments, the ligand includes an aromatic group. Methods, systems,and compositions of the present invention are not limited by the numberof different ligand types used. There may be 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more different types of ligands attached to a dendrimer molecule.

In some embodiments, conjugation between a ligand and a functional groupor between functional groups is accomplished through use of a1,3-dipolar cycloaddition reaction (“click chemistry”). ‘Clickchemistry’ involves, for example, the coupling of two different moieties(e.g., a therapeutic agent and a functional group) (e.g., a firstfunctional group and a second functional group) via a 1,3-dipolarcycloaddition reaction between an alkyne moiety (or equivalent thereof)on the surface of the first moeity and an azide moiety (or equivalentthereof) (or any active end group such as, for example, a primary amineend group, a hydroxyl end group, a carboxylic acid end group, a thiolend group, etc.) on the second moiety. ‘Click chemistry’ is anattractive coupling method because, for example, it can be performedwith a wide variety of solvent conditions including aqueousenvironments. For example, the stable triazole ring that results fromcoupling the alkyne with the azide is frequently achieved atquantitative yields and is considered to be biologically inert (see,e.g., Rostovtsev, V. V.; et al., Angewandte Chemie-International Edition2002, 41, (14), 2596; Wu, P.; et al., Angewandte Chemie-InternationalEdition 2004, 43, (30), 3928-3932; each herein incorporated by referencein their entireties).

The present invention is not limited to particular functional groups(e.g., for conjugation with dendrimers). Examples of functional groupsinclude but are not limited to therapeutic agents, targeting agents,trigger agents, and imaging agents.

In some embodiments, the functional group(s) is attached with thedendrimer via a linker. The present invention is not limited to aparticular type or kind of linker. In some embodiments, the linkercomprises a spacer comprising between 1 and 8 straight or branchedcarbon chains. In some embodiments, the straight or branched carbonchains are unsubstituted. In some embodiments, the straight or branchedcarbon chains are substituted with alkyls.

Examples of therapeutic agents include, but are not limited to, achemotherapeutic agent, an anti-oncogenic agent, an anti-angiogenicagent, a tumor suppressor agent, an anti-microbial agent, an expressionconstruct comprising a nucleic acid encoding a therapeutic protein, apain relief agent, a pain relief agent antagonist, an agent designed totreat an inflammatory disorder, an agent designed to treat an autoimmunedisorder, an agent designed to treat inflammatory bowel disease, and anagent designed to treat inflammatory pelvic disease. In someembodiments, the agent designed to treat an inflammatory disorderincludes, but is not limited to, an antirheumatic drug, a biologicalsagent, a nonsteroidal anti-inflammatory drug, an analgesic, animmunomodulator, a glucocorticoid, a TNF-α inhibitor, an IL-1 inhibitor,and a metalloprotease inhibitor. In some embodiments, the antirheumaticdrug includes, but is not limited to, leflunomide, methotrexate,sulfasalazine, and hydroxychloroquine. Examples of biologicals agentsinclude, but are not limited to, rituximab, finfliximab, etanercept,adalimumab, and golimumab. In some embodiments, the nonsteroidalanti-inflammatory drug includes, but is not limited to, ibuprofen,celecoxib, ketoprofen, naproxen, piroxicam, and diclofenac. In someembodiments, the analgesic includes, but is not limited to,acetaminophen, and tramadol. In some embodiments, the immunomodulatorincludes but is not limited to anakinra, and abatacept. In someembodiments, the glucocorticoid includes, but is not limited to,prednisone, and methylprednisone. In some embodiments, the TNF-αinhibitor includes but is not limited to adalimumab, certolizumab pegol,etanercept, golimumab, and infliximab. In some embodiments, theautoimmune disorder and/or inflammatory disorder includes, but is notlimited to, arthritis, psoriasis, lupus erythematosus, Crohn's disease,and sarcoidosis. In some embodiments, examples of arthritis include, butare not limited to, osteoarthritis, rheumatoid arthritis, septicarthritis, gout and pseudo-gout, juvenile idiopathic arthritis,psoriatic arthritis, Still's disease, and ankylosing spondylitis.

Ligands suitable for use in certain method embodiments of the presentinvention are not limited to a particular type or kind of targetingagent. In some embodiments, the targeting agent is configured to targetthe composition to cancer cells. In some embodiments, the targetingagent comprises FA. In some embodiments, the targeting agent binds areceptor selected from the group consisting of CFTR, EGFR, estrogenreceptor, FGR2, folate receptor, IL-2 receptor, and VEGFR. In someembodiments, the targeting agent comprises an antibody that binds to apolypeptide selected from the group consisting of p53, Muc1, a mutatedversion of p53 that is present in breast cancer, HER-2, T and Tn haptensin glycoproteins of human breast carcinoma, and MSA breast carcinomaglycoprotein. In some embodiments, the targeting agent comprises anantibody selected from the group consisting of human carcinoma antigen,TP1 and TP3 antigens from osteocarcinoma cells, Thomsen-Friedenreich(TF) antigen from adenocarcinoma cells, KC-4 antigen from humanprostrate adenocarcinoma, human colorectal cancer antigen, CA125 antigenfrom cystadenocarcinoma, DF3 antigen from human breast carcinoma, andp97 antigen of human melanoma, carcinoma or orosomucoid-related antigen.In some embodiments, the targeting agent is configured to permit thecomposition to cross the blood brain barrier. In some embodiments, thetargeting agent is transferrin. In some embodiments, the targeting agentis configured to permit the composition to bind with a neuron within thecentral nervous system. In some embodiments, the targeting agent is asynthetic tetanus toxin fragment. In some embodiments, the synthetictetanus toxin fragment comprises an amino acid peptide fragment. In someembodiments, the amino acid peptide fragment is HLNILSTLWKYR (SEQ ID NO:2).

In some embodiments, the ligand comprises a trigger agent. The presentinvention is not limited to particular type or kind of trigger agent. Insome embodiments, the trigger agent is configured to have a functionsuch as, for example, a) a delayed release of a functional group fromthe dendrimer, b) a constitutive release of the therapeutic agent fromthe dendrimer, c) a release of a functional group from the dendrimerunder conditions of acidosis, d) a release of a functional group from adendrimer under conditions of hypoxia, and e) a release of thetherapeutic agent from a dendrimer in the presence of a brain enzyme.Examples of trigger agents include, but are not limited to, an esterbond, an amide bond, an ether bond, an indoquinone, a nitroheterocyle,and a nitroimidazole.

Ligands suitable for use in certain method embodiments of the presentinvention are not limited to a particular type or kind of imaging agent.Examples of imaging agents include, but are not limited to, fluoresceinisothiocyanate (FITC), 6-TAMARA, acridine orange, and cis-parinaricacid.

In certain embodiments, the present invention provides a compositioncomprising a PAMAM dendrimer conjugated with a triazine compound. Insome embodiments, the PAMAM dendrimer is conjugated with the triazinecompound via a 3-azido-coumarine linkage agent. In some embodiments, thetriazine compound comprises a 1,3,5-triazine compound. In someembodiments, the triazine compound is trivalent. In some embodiments,the triazine compound is conjugated with one or more ligands. In someembodiments, the one or more ligands are independently selected fromtypes such as a therapeutic agent, a targeting agent, an imaging agent,and a trigger agent. In some embodiments, the one or more ligands areMTX, FA, and 3-azido-coumarine. In some embodiments, the triazinecompound is configured to be compatible with click chemistry. In someembodiments, the triazine compound comprises an azido group. In someembodiments, the triazine compound is a compound such as

In certain embodiments, the present invention provides a method ofsynthesizing a functionalized dendrimer, the method comprising: a)providing an alkyne-modified dendrimer (e.g., PAMAM dendrimer) and atriazine compound; and b) conjugating the alkyne-modified dendrimer withthe triazine compound. In some embodiments, the conjugating of thealkyne-modified dendrimer (e.g., PAMAM dendrimer) with the triazinecompound occurs via a click chemistry reaction. In some embodiments, thedendrimer (e.g., Baker-Huang PAMAM dendrimer; PAMAM dendrimer) isconjugated with the triazine compound via a 3-azido-coumarine linkageagent. In some embodiments, the method further comprises: step c)conjugating one or more ligands with the triazine compound. In someembodiments, the one or more ligands are types such as a therapeuticagent, a targeting agent, an imaging agent, and a trigger agent. In someembodiments, the one or more ligands are MTX and FA. In someembodiments, the triazine compound comprises an azido group.

In certain embodiments, the present invention provides methods fortreating a disorder selected from the group consisting of any type ofcancer or cancer-related disorder (e.g., tumor, a neoplasm, a lymphoma,or a leukemia), a neoplastic disease, osteoarthritis, rheumatoidarthritis, septic arthritis, gout and pseudo-gout, juvenile idiopathicarthritis, psoriatic arthritis, Still's disease, and ankylosingspondylitis, comprising administering to a subject suffering from thedisorder a dendrimer generated with the methods of the presentinvention. In some embodiments, the dendrimer is co-administered with anadditional agent(s) so as to enhance such a treatment.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows molecular structures of triazines 6 and 9.

FIG. 2 shows one-step surface modification of the G5 PAMAM dendrimer byclick chemistry.

FIG. 3 shows a synthetic scheme of triazine 6.

FIG. 4 shows a synthetic scheme of triazine 11.

FIG. 5 shows a synthetic scheme of Triazine-N₃-γMTX-αFA 8.

FIG. 6 shows synthesis of γMTX-ethylamine 11.

FIG. 7 shows a synthetic scheme of Triazine-N₃-γMTX-NHBoc 13.

FIG. 8 shows synthesis of dendrimer conjugates G5-Triazine-γMTX-αFA 15,G5-Triazine-αFA 16, and G5-Triazine-γMTX 17.

FIG. 9 shows cytotoxicity of the triazine-linked G5-conjugatesG5-Triazine-αFA 16, G5-Triazine-γMTX 17 and G5-Triazine-γMTX-αFA 15. KBcells were incubated for 48 h with the indicated conjugates, the mediumand drugs were then replaced with fresh medium and drugs and incubatedfor additional 48 h. The cytotoxicity was determined by XTT assay asgiven in the text. Also shown is the cytotoxicity of a previous batch ofG5-FA-MTX (CMBX 123-34) containing average 4-5 FA and 7-8 MTX perdendrimer.

FIG. 10 shows release of free MTX and FA from G5-Triazine-γMTX-αFAconjugate 15 in cell culture medium. 15 (100 μM) was incubated with RPMImedium containing final 10% serum for different time periods and themedium proteins were precipitated with 10% DMSO in acetonitrile. Thesupernatant obtained were subjected HPLC analysis. The inset shows theelution peak areas of the FA and MTX. The values given on the Y-axis areexpressed as the percent mole of total MTX present per mole of thedendrimer.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “subject suspected of having cancer” refers toa subject that presents one or more symptoms indicative of a cancer(e.g., a noticeable lump or mass) or is being screened for a cancer(e.g., during a routine physical). A subject suspected of having cancermay also have one or more risk factors. A subject suspected of havingcancer has generally not been tested for cancer. However, a “subjectsuspected of having cancer” encompasses an individual who has received apreliminary diagnosis (e.g., a CT scan showing a mass) but for whom aconfirmatory test (e.g., biopsy and/or histology) has not been done orfor whom the stage of cancer is not known. The term further includespeople who once had cancer (e.g., an individual in remission). A“subject suspected of having cancer” is sometimes diagnosed with cancerand is sometimes found to not have cancer.

As used herein, the term “subject diagnosed with a cancer” refers to asubject who has been tested and found to have cancerous cells. Thecancer may be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention.

As used herein, the term “initial diagnosis” refers to a test result ofinitial cancer diagnosis that reveals the presence or absence ofcancerous cells (e.g., using a biopsy and histology).

As used herein, the term “identifying the risk of said tumormetastasizing” refers to the relative risk (e.g., the percent chance ora relative score) of a tumor metastasizing.

As used herein, the term “identifying the risk of said tumor recurring”refers to the relative risk (e.g., the percent chance or a relativescore) of a tumor recurring in the same organ as the original tumor.

As used herein, the term “subject at risk for cancer” refers to asubject with one or more risk factors for developing a specific cancer.Risk factors include, but are not limited to, gender, age, geneticpredisposition, environmental expose, and previous incidents of cancer,preexisting non-cancer diseases, and lifestyle.

As used herein, the term “characterizing cancer in subject” refers tothe identification of one or more properties of a cancer sample in asubject, including but not limited to, the presence of benign,pre-cancerous or cancerous tissue and the stage of the cancer.

As used herein, the term “stage of cancer” refers to a qualitative orquantitative assessment of the level of advancement of a cancer.Criteria used to determine the stage of a cancer include, but are notlimited to, the size of the tumor, whether the tumor has spread to otherparts of the body and where the cancer has spread (e.g., within the sameorgan or region of the body or to another organ).

As used herein, the term “providing a prognosis” refers to providinginformation regarding the impact of the presence of cancer on asubject's future health (e.g., expected morbidity or mortality, thelikelihood of getting cancer, and the risk of metastasis).

As used herein, the term “non-human animals” refers to all non-humananimals including, but not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, ayes, etc.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the presentinvention.

As used herein, the term “drug” is meant to include any molecule,molecular complex or substance administered to an organism fordiagnostic or therapeutic purposes, including medical imaging,monitoring, contraceptive, cosmetic, nutraceutical, pharmaceutical andprophylactic applications. The term “drug” is further meant to includeany such molecule, molecular complex or substance that is chemicallymodified and/or operatively attached to a biologic or biocompatiblestructure.

As used herein, the term “purified” or “to purify” or “compositionalpurity” refers to the removal of components (e.g., contaminants) from asample or the level of components (e.g., contaminants) within a sample.For example, unreacted moieties, degradation products, excess reactants,or byproducts are removed from a sample following a synthesis reactionor preparative method.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness, sickness, or disorder of bodilyfunction (e.g., cancer). Test compounds comprise both known andpotential therapeutic compounds. A test compound can be determined to betherapeutic by screening using screening methods known in the art.

As used herein, the term “nanodevice” or “nanodevices” refer, generally,to compositions comprising dendrimers of the present invention. As such,a nanodevice may refer to a composition comprising a dendrimer of thepresent invention that may contain one or more ligands, linkers, and/orfunctional groups (e.g., a therapeutic agent, a targeting agent, atrigger agent, an imaging agent) conjugated to the dendrimer.

As used herein, the term “degradable linkage,” when used in reference toa polymer refers to a conjugate that comprises a physiologicallycleavable linkage (e.g., a linkage that can be hydrolyzed (e.g., invivo) or otherwise reversed (e.g., via enzymatic cleavage). Suchphysiologically cleavable linkages include, but are not limited to,ester, carbonate ester, carbamate, sulfate, phosphate, acyloxyalkylether, acetal, and ketal linkages (See, e.g., U.S. Pat. No. 6,838,076,herein incorporated by reference in its entirety). Similarly, theconjugate may comprise a cleavable linkage present in the linkagebetween the dendrimer and functional group, or, may comprise a cleavablelinkage present in the polymer itself (See, e.g., U.S. Pat. App. Nos.20050158273 and 20050181449, each of which is herein incorporated byreference in its entirety).

A “physiologically cleavable” or “hydrolysable” or “degradable” bond isa bond that reacts with water (i.e., is hydrolyzed) under physiologicalconditions. The tendency of a bond to hydrolyze in water will depend notonly on the general type of linkage connecting two central atoms butalso on the substituents attached to these central atoms. Appropriatehydrolytically unstable or weak linkages include but are not limited tocarboxylate ester, phosphate ester, anhydrides, acetals, ketals,acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond(e.g., typically a covalent bond) that is substantially stable in water(i.e., does not undergo hydrolysis under physiological conditions to anyappreciable extent over an extended period of time). Examples ofhydrolytically stable linkages include, but are not limited to,carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides,urethanes, and the like.

As used herein, the term “NAALADase inhibitor” refers to any one of amultitude of inhibitors for the neuropeptidase NAALADase(N-acetylated-alpha linked acidic dipeptidase). Such inhibitors ofNAALADase have been well characterized. For example, an inhibitor can beselected from the group comprising, but not limited to, those found inU.S. Pat. No. 6,011,021, herein incorporated by reference in itsentirety.

As used herein, the term “Baker-Huang dendrimer” or “Baker-Huang PAMAMdendrimer” refers to a dendrimer comprised of branching units ofstructure:

wherein R comprises a carbon-containing functional group (e.g., CF₃). Insome embodiments, the branching unit is activated to its HNS ester. Insome embodiments, such activation is achieved using TSTU. In someembodiments, EDA is added. In some embodiments, the dendrimer is furthertreated to replace, e.g., CF₃ functional groups with NH₂ functionalgroups; for example, in some embodiments, a CF₃-containing version ofthe dendrimer is treated with K₂CO₃ to yield a dendrimer with terminalNH₂ groups (for example, as shown in Scheme 2). In some embodiments,terminal groups of a Baker-Huang dendrimer are further derivatizedand/or further conjugated with other moieties. For example, one or morefunctional ligands (e.g., for therapeutic, targeting, imaging, or drugdelivery function(s)) may be conjugated to a Baker-Huang dendrimer,either via direct conjugation to terminal branches or indirectly (e.g.,through linkers, through other functional groups (e.g., through an OH—functional group)). In some embodiments, the order of iterative repeatsfrom core to surface is amide bonds first, followed by tertiary amines,with ethylene groups intervening between the amide bond and tertiaryamines. In preferred embodiments, a Baker-Huang dendrimer is synthesizedby convergent synthesis methods.

As used herein, the term “click chemistry” refers to chemistry tailoredto generate substances quickly and reliably by joining small modularunits together (see, e.g., Kolb et al. (2001) Angewandte Chemie Intl.Ed. 40:2004-2011; Evans (2007) Australian J. Chem. 60:384-395; Carlmarket al. (2009) Chem. Soc. Rev. 38:352-362; each herein incorporated byreference in its entirety).

As used herein, the term “triazine” refers to a compound comprising aring structure bearing three nitrogen atoms. In some embodiments, thering structure is six-membered (e.g., the molecular formula comprisesC₃H₃N₃). In some embodiments, the ring is a conjugated system. Triazinemoieties with six-membered rings may have nitrogen atoms at any possibleplacement so long as three nitrogen atoms occur in the ring (e.g.,1,2,3-triazine; 1,2,4-triazine, 1,3,5-triazine, 1,2,5-triazine,1,2,6-triazine, etc.)

As used herein, the term “scaffold” refers to a compound to which othermoieties are attached (e.g., conjugated). In some embodiments, ascaffold is conjugated to bioactive functional conjugates (e.g., atherapeutic agent, a targeting agent, a trigger agent, an imagingagent). In some embodiments, a scaffold is conjugated to a dendrimer(e.g., a PAMAM dendrimer). In some embodiments, conjugation of ascaffold to a dendrimer and/or a functional conjugate(s) is direct,while in other embodiments conjugation of a scaffold to a dendrimerand/or a functional conjugate(s) is indirect, e.g., an interveninglinker is present between the scaffold compound and the dendrimer,and/or the scaffold and the functional conjugate(s).

As used herein, the term “one-pot synthesis reaction” or equivalentsthereof, e.g., “1-pot”, “one pot”, etc., refers to a chemical synthesismethod in which all reactants are present in a single vessel. Reactantsmay be added simultaneously or sequentially, with no limitation as tothe duration of time elapsing between introduction of sequentially addedreactants. In some embodiments, conjugation between a dendrimer (e.g., aterminal arm of a dendrimer) and a functional ligand is accomplishedduring a “one-pot” reaction. In some embodiments, a one-pot reactionoccurs wherein a hydroxyl-terminated dendrimer (e.g., HO-PAMAMdendrimer) is reacted with one or more functional ligands (e.g., atherapeutic agent, a pro-drug, a trigger agent, a targeting agent, animaging agent) in one vessel, such conjugation being facilitated byester coupling agents (e.g., 2-chloro-1-methylpyridinium iodide and4-(dimethylamino) pyridine) (see, e.g., International Patent ApplicationNo. PCT/US2010/042556, herein incorporated by reference in itsentirety).

As used herein, the term “solvent” refers to a medium in which areaction is conducted. Solvents may be liquid but are not limited toliquid form. Solvent categories include but are not limited to nonpolar,polar, protic, and aprotic.

As used herein, the term “dialysis” refers to a purification method inwhich the solution surrounding a substance is exchanged over time withanother solution. Dialysis is generally performed in liquid phase byplacing a sample in a chamber, tubing, or other device with aselectively permeable membrane. In some embodiments, the selectivelypermeable membrane is cellulose membrane. In some embodiments, dialysisis performed for the purpose of buffer exchange. In some embodiments,dialysis may achieve concentration of the original sample volume. Insome embodiments, dialysis may achieve dilution of the original samplevolume.

As used herein, the term “precipitation” refers to purification of asubstance by causing it to take solid form, usually within a liquidcontext. Precipitation may then allow collection of the purifiedsubstance by physical handling, e.g. centrifugation or filtration.

As used herein, an “ester coupling agent” refers to a reagent that canfacilitate the formation of an ester bond between two reactants. Thepresent invention is not limited to any particular coupling agent oragents. Examples of coupling agents include but are not limited to2-chloro-1-methylpyridium iodide and 4-(dimethylamino) pyridine, ordicyclohexylcarbodiimide and 4-(dimethylamino) pyridine or diethylazodicarboxylate and triphenylphosphine or other carbodiimide couplingagent and 4-(dimethylamino)pyridine.

As used herein, the term “glycidolate” refers to the addition of a2,3-dihydroxylpropyl group to a reagent using glycidol as a reactant. Insome embodiments, the reagent to which the 2,3-dihydroxylpropyl groupsare added is a dendrimer. In some embodiments, the dendrimer is a PAMAMdendrimer. Glycidolation may be used generally to add terminal hydroxylfunctional groups to a reagent.

As used herein, the term “ligand” refers to any moiety covalentlyattached (e.g., conjugated) to a dendrimer branch; in preferredembodiments, such conjugation is indirect (e.g., an intervening moietyexists between the dendrimer branch and the ligand) rather than direct(e.g., no intervening moiety exists between the dendrimer branch and theligand). Indirect attachment of a ligand to a dendrimer may exist wherea scaffold compound (e.g., triazine scaffold) intervenes. In preferredembodiments, ligands have functional utility for specific applications,e.g., for therapeutic, targeting, imaging, or drug delivery function(s).The terms “ligand”, “conjugate”, and “functional group” may be usedinterchangeably.

DETAILED DESCRIPTION OF THE INVENTION

Polymers have been used as drug carriers due to their ability to releasedrugs in a controlled manner, prolong circulation times, and increasedrug solubility. Major efforts have been made to synthesize functionalpolymers that lead to the components of multipurpose therapeutic ordiagnostic materials. Because of their well-defined structures withprecise control of the size and shape as well as terminal functionalgroups, PAMAM dendrimers are well suited to serve as a platform for thedevelopment of an effective tumor targeting drug delivery device (Tekadeet al. (2009) Chem. Rev. 109:49-87; Landers et al. (2002) J. Infect.Dis. 186:1222-1230; Weiner et al. (1997) Invest. Radiol. 32:748-754;Delong et al. (1997) J. Pharm. Sci. 86:762-764; Gillies et al. (2005)Drug Discovery Today 10:35-43; each herein incorporated by reference inits entirety). A multi-step synthetic procedure is commonly employed tosynthesize the multifunctional dendrimers (Kukowska-Latallo et al.(2005) Canc. Res. 65:5317-5324; Thomas et al. (2005) J. Med. Chem.48:3729-3735; Quintana et al. (2002) Pharmaceu. Res. 19:1310-1316; eachherein incorporated by reference in its entirety). However, multi-stepprocedures introduce higher polydispersity, which has limited theirapplication. Moreover, it is extremely difficult to preciselycharacterize the attached components after each procedure. In someembodiments, methods of the present invention provide conjugation ofdendrimers with multifunctional small molecule architectures, which areloaded with different bioactive molecules. These small moleculederivatives can be easily purified and characterized before attachmentto the dendrimers.

In experiments conducted during the course of developing someembodiments of the present invention, a unique structural class 6 and 11was developed (FIG. 1). Specifically, in some embodiments, a dendriticstructure was developed which integrates bioactive molecules (e.g., FAand MTX) and a fluorophore (for 11). In these new multifunctionalmolecules, the trifunctional triazine was chosen as a core scaffold onwhich two sites could be used for binding bioactive molecules while anazide linker could be linked to its third site. High-efficiency ‘clickchemistry’ was employed for the one-step conjugation to the alkyneterminal dendrimer (FIG. 2), which reduced the cross-linking byproductsfrom typical coupling reactions (Kolb et al. (2001) AngewandteChemie-Intl. Ed. 40:2004-2021; Kolb et al. (2003) Drug Disc. Today8:1128-1137; Lewis et al. (2002) Angewandte Chemie-Intl. Ed.41:1053-1057; each herein incorporated by reference in its entirety).The triazine derivatives were synthesized from cyanuric chloride byconsecutive aromatic nucleophilic substitution reactions undercontrolled conditions. Coumarine was chosen as the fluorophore due toits small size, biocompatibility, and ease of to synthetic manipulation(Sivakumar et al. (2004) Organic Lett. 6:4603-4606; herein incorporatedby reference in its entirety).

Advantages of choosing triazines as substrates to build multivalentPAMAM dendrimers include but are not limited to 1) the fact thatmultifunctional dendrimers with targeting moiety, drug, and imagingreagent can be achieved by one-step dendrimer modification, leading to anarrow range of dendrimer molecular weights, and 2) the fact that thesequential reactivity of the three chlorine atoms in the triazinebackbone makes these molecules well-suited to get high variability,useful for combinatorial synthesis. Methods and compositions of someembodiments of the present invention provide access to a novel syntheticroute to multifunctional dendrimers.

Moreover, experiments conducted during the course of developing someembodiments of the present invention demonstrated that such derivatives(e.g., triazine derivatives incorporating the targeting moiety FA andtherapeutic drug MTX conjugated to PAMAM dendrimers through “clickchemistry”) demonstrated an in vitro dose-dependent cytotoxicity withhigher cytotoxic potential vs. a similar conjugate synthesized by amulti-step approach (see, e.g., FIGS. 9 and 10 and Example III).

In some embodiments, the present invention provides compositionsfacilitating one-step (e.g., “click chemistry”) conjugation offunctional groups to dendrimers (e.g., terminal arms of dendrimers). Insome embodiments, such compositions comprise multifunctional smallmolecule architectures (e.g., scaffolds) which permit conjugation tofunctional groups (e.g., therapeutic groups, imaging groups, targetinggroups, pro-drugs complexes, trigger groups). In some embodiments, suchfunctional group-conjugated compositions are used for one-stepconjugation to dendrimers (e.g., to terminal branches of dendrimers ormodified dendrimers). In some embodiments, compositions of the presentinvention comprise triazine compositions. In some embodiments, atriazine composition is trifunctional such that two sites are used forconjugation or binding to functional groups (e.g., bioactive molecules)and an azide linker is conjugated (e.g., linked) to the third site.Compositions of the present invention are not limited by the numericalfunctionality, e.g., bifunctional, trifunctional, quadfunctional orembodiments with higher degrees of are contemplated.

The present invention is not limited to the use of particular typesand/or kinds of dendrimers (e.g., a dendrimer conjugated with at leastone functional group). Indeed, dendrimeric polymers have been describedextensively (See, e.g., Tomalia, Advanced Materials 6:529 (1994); Angew,Chem. Int. Ed. Engl., 29:138 (1990); incorporated herein by reference intheir entireties). Dendrimer polymers are synthesized as definedspherical structures typically ranging from 1 to 20 nanometers indiameter. Methods for manufacturing a G5 PAMAM dendrimer with aprotected core are known (U.S. patent application Ser. No. 12/403,179;herein incorporated by reference in its entirety). In preferredembodiments, the protected core diamine is NH₂—CH₂—CH₂—NHPG. Molecularweight and the number of terminal groups increase exponentially as afunction of generation (the number of layers) of the polymer. In someembodiments of the present invention, half generation PAMAM dendrimersare used. For example, when an ethylenediamine (EDA) core is used fordendrimer synthesis, alkylation of this core through Michael additionresults in a half-generation molecule with ester terminal groups;amidation of such ester groups with excess EDA results in creation of afull-generation, amine-terminated dendrimer (Majoros et al., Eds. (2008)Dendrimer-based Nanomedicine, Pan Stanford Publishing Pte. Ltd.,Singapore, p. 42). Different types of dendrimers can be synthesizedbased on the core structure that initiates the polymerization process.In some embodiments, the PAMAM dendrimers are “Baker-Huang dendrimers”or “Baker-Huang PAMAM dendrimers” (see, e.g., U.S. Provisional PatentApplication Ser. No. 61/251,244; herein incorporated by reference in itsentirety).

The dendrimer core structures dictate several characteristics of themolecule such as the overall shape, density and surface functionality(See, e.g., Tomalia et al., Chem. Int. Ed. Engl., 29:5305 (1990)).Spherical dendrimers can have ammonia as a trivalent initiator core orethylenediamine (EDA) as a tetravalent initiator core. Recentlydescribed rod-shaped dendrimers (See, e.g., Yin et al., J. Am. Chem.Soc., 120:2678 (1998)) use polyethyleneimine linear cores of varyinglengths; the longer the core, the longer the rod. Dendriticmacromolecules are available commercially in kilogram quantities and areproduced under current good manufacturing processes (GMP) forbiotechnology applications.

Dendrimers may be characterized by a number of techniques including, butnot limited to, electrospray-ionization mass spectroscopy, ¹³C nuclearmagnetic resonance spectroscopy, ¹H nuclear magnetic resonancespectroscopy, size exclusion chromatography with multi-angle laser lightscattering, ultraviolet spectrophotometry, capillary electrophoresis andgel electrophoresis. These tests assure the uniformity of the polymerpopulation and are important for monitoring quality control of dendrimermanufacture for GMP applications and in vivo usage.

Numerous U.S. Patents describe methods and compositions for producingdendrimers. Examples of some of these patents are given below in orderto provide a description of some dendrimer compositions that may beuseful in the present invention, however it should be understood thatthese are merely illustrative examples and numerous other similardendrimer compositions could be used in the present invention.

U.S. Pat. No. 4,507,466, U.S. Pat. No. 4,558,120, U.S. Pat. No.4,568,737, and U.S. Pat. No. 4,587,329 each describes methods of makingdense star polymers with terminal densities greater than conventionalstar polymers. These polymers have greater/more uniform reactivity thanconventional star polymers, i.e. 3rd generation dense star polymers.These patents further describe the nature of the amidoamine dendrimersand the 3-dimensional molecular diameter of the dendrimers.

U.S. Pat. No. 4,631,337 describes hydrolytically stable polymers. U.S.Pat. No. 4,694,064 describes rod-shaped dendrimers. U.S. Pat. No.4,713,975 describes dense star polymers and their use to characterizesurfaces of viruses, bacteria and proteins including enzymes. Bridgeddense star polymers are described in U.S. Pat. No. 4,737,550. U.S. Pat.No. 4,857,599 and U.S. Pat. No. 4,871,779 describe dense star polymerson immobilized cores useful as ion-exchange resins, chelation resins andmethods of making such polymers.

U.S. Pat. No. 5,338,532 is directed to starburst conjugates ofdendrimer(s) in association with at least one unit of carriedagricultural, pharmaceutical or other material. This patent describesthe use of dendrimers to provide means of delivery of highconcentrations of carried materials per unit polymer, controlleddelivery, targeted delivery and/or multiple species such as e.g., drugsantibiotics, general and specific toxins, metal ions, radionuclides,signal generators, antibodies, interleukins, hormones, interferons,viruses, viral fragments, pesticides, and antimicrobials.

U.S. Pat. No. 6,471,968 describes a dendrimer complex comprisingcovalently linked first and second dendrimers, with the first dendrimercomprising a first agent and the second dendrimer comprising a secondagent, wherein the first dendrimer is different from the seconddendrimer, and where the first agent is different than the second agent.

Other useful dendrimer type compositions are described in U.S. Pat. No.5,387,617, U.S. Pat. No. 5,393,797, and U.S. Pat. No. 5,393,795 in whichdense star polymers are modified by capping with a hydrophobic groupcapable of providing a hydrophobic outer shell. U.S. Pat. No. 5,527,524discloses the use of amino terminated dendrimers in antibody conjugates.

PAMAM dendrimers are highly branched, narrowly dispersed syntheticmacromolecules with well-defined chemical structures.

PAMAM dendrimers can be easily modified and conjugated with multiplefunctionalities such as targeting molecules, imaging agents, and drugs(Thomas et al. (2007) Poly(amidoamine) Dendrimer-based MultifunctionalNanoparticles, in Nanobiotechnology: Concepts, Methods and Perspectives,Merkin, Ed., Wiley-VCH; herein incorporated by reference in itsentirety). They are water soluble, biocompatible, and cleared from theblood through the kidneys (Peer et al. (2007) Nat. Nanotechnol.2:751-760; herein incorporated by reference in its entirety) whicheliminates the need for biodegradability. Because of these desirableproperties, PAMAM dendrimers have been widely investigated for drugdelivery (Esfand et al. (2001) Drug Discov. Today 6:427-436; Patri etal. (2002) Curr. Opin. Chem. Biol. 6:466-471; Kukowska-Latallo et al.(2005) Cancer Res. 65:5317-5324; Quintana et al. (2002) PharmaceuticalRes. 19:1310-1316; Thomas et al. (2005) J. Med. Chem. 48:3729-3735; eachherein incorporated by reference in its entirety), gene therapy(KukowskaLatallo et al. (1996) PNAS 93:4897-4902; Eichman et al. (2000)Pharm. Sci. Technolo. Today 3:232-245; Luo et al. (2002) Macromol.35:3456-3462; each herein incorporated by reference in its entirety),and imaging applications (Kobayashi et al. (2003) Bioconj. Chem.14:388-394; herein incorporated by reference in its entirety).

The use of dendrimers as metal ion carriers is described in U.S. Pat.No. 5,560,929. U.S. Pat. No. 5,773,527 discloses non-crosslinkedpolybranched polymers having a comb-burst configuration and methods ofmaking the same. U.S. Pat. No. 5,631,329 describes a process to producepolybranched polymer of high molecular weight by forming a first set ofbranched polymers protected from branching; grafting to a core;deprotecting first set branched polymer, then forming a second set ofbranched polymers protected from branching and grafting to the corehaving the first set of branched polymers, etc.

U.S. Pat. No. 5,902,863 describes dendrimer networks containinglipophilic organosilicone and hydrophilic polyanicloamine nanscopicdomains. The networks are prepared from copolydendrimer precursorshaving PAMAM (hydrophilic) or polyproyleneimine interiors andorganosilicon outer layers. These dendrimers have a controllable size,shape and spatial distribution. They are hydrophobic dendrimers with anorganosilicon outer layer that can be used for specialty membrane,protective coating, composites containing organic organometallic orinorganic additives, skin patch delivery, absorbants, chromatographypersonal care products and agricultural products.

U.S. Pat. No. 5,795,582 describes the use of dendrimers as adjuvants forinfluenza antigen. Use of the dendrimers produces antibody titer levelswith reduced antigen dose. U.S. Pat. No. 5,898,005 and U.S. Pat. No.5,861,319 describe specific immunobinding assays for determiningconcentration of an analyte. U.S. Pat. No. 5,661,025 provides details ofa self-assembling polynucleotide delivery system comprising dendrimerpolycation to aid in delivery of nucleotides to target site. This patentprovides methods of introducing a polynucleotide into a eukaryotic cellin vitro comprising contacting the cell with a composition comprising apolynucleotide and a dendrimer polyeation non-covalently coupled to thepolynucleotide.

Dendrimer-antibody conjugates for use in in vitro diagnosticapplications have previously been demonstrated (See, e.g., Singh et al.,Clin. Chem., 40:1845 (1994)), for the production ofdendrimer-chelant-antibody constructs, and for the development ofboronated dendrimer-antibody conjugates (for neutron capture therapy);each of these latter compounds may be used as a cancer therapeutic (See,e.g., Wu et al., Bioorg. Med. Chem. Lett., 4:449 (1994); Wiener et al.,Magn. Reson. Med. 31:1 (1994); Barth et al., Bioconjugate Chem. 5:58(1994); and Barth et al.).

Some of these conjugates have also been employed in the magneticresonance imaging of tumors (See, e.g., Wu et al., (1994) and Wiener etal., (1994), supra). Results from this work have documented that, whenadministered in vivo, antibodies can direct dendrimer-associatedtherapeutic agents to antigen-bearing tumors. Dendrimers also have beenshown to specifically enter cells and carry either chemotherapeuticagents or genetic therapeutics. In particular, studies show thatcisplatin encapsulated in dendrimer polymers has increased efficacy andis less toxic than cisplatin delivered by other means (See, e.g., Duncanand Malik, Control Rel. Bioact. Mater. 23:105 (1996)).

Dendrimers have also been conjugated to fluorochromes or molecularbeacons and shown to enter cells. They can then be detected within thecell in a manner compatible with sensing apparatus for evaluation ofphysiologic changes within cells (See, e.g., Baker et al., Anal. Chem.69:990 (1997)). Finally, dendrimers have been constructed asdifferentiated block copolymers where the outer portions of the moleculemay be digested with either enzyme or light-induced catalysis (See,e.g., Urdea and Hom, Science 261:534 (1993)). This allows the controlleddegradation of the polymer to release therapeutics at the disease siteand provides a mechanism for an external trigger to release thetherapeutic agents.

In experiments conducted during the course of developing embodiments forthe present invention, dendrimers containing functional components weredeveloped having a therapeutic agent (e.g., a chemotherapeutic agent)conjugated with one or more functional groups (e.g., an imaging agent)and a targeting agent (e.g., folic acid)). The present invention iscompatible with additional functional components (e.g., trigger agents(e.g., for release under hypoxic conditions)). In some embodiments,synthesis of the functionalized dendrimer is facilitated by use oftriazine molecules that are linked to functional components and used forone-step (e.g., click chemistry) addition to terminal arms ofdendrimers.

Accordingly, the present invention provides synthesis methods,compositions and applications for efficient, site-specific drug deliveryusing functionalized dendrimers comprising therapeutic agents conjugatedwith one or more functional groups (e.g., imaging agents, targetingagents, and trigger agents). In particular, the present inventionrelates to dendrimers comprising one or more functional groupsconjugated with a therapeutic agent (e.g., a chemotherapeutic agent),methods of synthesizing the same, as well as systems and methodsutilizing the therapeutic and diagnostic compositions (e.g., indiagnostic and/or therapeutic settings (e.g., for the delivery oftherapeutics, imaging, and/or targeting agents (e.g., in disease (e.g.,cancer) diagnosis and/or therapy, etc.)). For example, in someembodiments, the novel therapeutic and diagnostic compositions comprisea therapeutic agent (e.g., a chemotherapeutic agent (e.g., MTX)conjugated with an imaging agent and a targeting agent (e.g., FA). Asdescribed in more detail below, examples of functional groups include,but are not limited to, targeting groups, trigger groups, and imaginggroups.

The present invention is not limited to the use of particulartherapeutic agents. In some embodiments, the therapeutic agents areeffective in treating autoimmune disorders and/or inflammatory disorders(e.g., arthritis). Examples of such therapeutic agents include, but arenot limited to, disease-modifying antirheumatic drugs (e.g.,leflunomide, methotrexate, sulfasalazine, hydroxychloroquine), biologicagents (e.g., rituximab, infliximab, etanercept, adalimumab, golimumab),nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, celecoxib,ketoprofen, naproxen, piroxicam, diclofenac), analgesics (e.g.,acetaminophen, tramadol), immunomodulators (e.g., anakinra, abatacept),glucocorticoids (e.g., prednisone, methylprednisone), TNF-α inhibitors(e.g., adalimumab, certolizumab pegol, etanercept, golimumab,infliximab), IL-1 inhibitors, and metalloprotease inhibitors. In someembodiments, the therapeutic agents include, but are not limited to,infliximab, adalimumab, etanercept, parenteral gold or oral gold.

In some embodiments, the therapeutic agents are effective in treatingcancer (see, e.g., U.S. Pat. Nos. 6,471,968, 7,078,461, and U.S. patentapplication Ser. Nos. 09/940,243, 10/431,682, 11/503,742, 11/661,465,11/523,509, 12/403,179, 12/106,876, and 11/827,637; and U.S. ProvisionalPatent Application Ser. Nos. 61/256,759, 61/140,840, 61/091,608,61/097,780, 61/101,461, 61/237,172, 61/229,168, 61/221,596, and61/251,244; each herein incorporated by reference in their entireties).

In some embodiments, the ligand (e.g., therapeutic agent) is conjugatedwith the dendrimer and/or triazine compound via a trigger agent. Thepresent invention is not limited to particular types or kinds of triggeragents.

In some embodiments, sustained release (e.g., slow release over a periodof 24-48 hours) of the ligand (e.g., therapeutic agent) is accomplishedthrough conjugating the therapeutic agent (e.g., directly) (e.g.,indirectly through one or more additional functional groups) to atrigger agent that slowly degrades in a biological system (e.g., amidelinkage, ester linkage, ether linkage). In some embodiments,constitutively active release of a therapeutic agent is accomplishedthrough conjugating the therapeutic agent to a trigger agent thatrenders the therapeutic agent constitutively active in a biologicalsystem (e.g., amide linkage, ether linkage).

In some embodiments, release of a therapeutic agent under specificconditions is accomplished through conjugating the therapeutic agent(e.g., directly) (e.g., indirectly through one or more additionalfunctional groups) to a trigger agent that degrades under such specificconditions (e.g., through activation of a trigger molecule underspecific conditions that leads to release of the therapeutic agent). Forexample, once a conjugate (e.g., a therapeutic agent conjugated with atrigger agent and a targeting agent) arrives at a target site in asubject (e.g., a tumor, or a site of inflammation), components in thetarget site (e.g., a tumor associated factor, or an inflammatory or painassociated factor) interact with the trigger agent thereby initiatingcleavage of the therapeutic agent from the trigger agent. In someembodiments, the trigger agent is configured to degrade (e.g., releasethe therapeutic agent) upon exposure to a tumor-associated factor (e.g.,hypoxia and pH, an enzyme (e.g., glucuronidase and/or plasmin), acathepsin, a matrix metalloproteinase, a hormone receptor (e.g.,integrin receptor, hyaluronic acid receptor, luteinizinghormone-releasing hormone receptor, etc.), cancer and/or tumor specificDNA sequence), an inflammatory associated factor (e.g., chemokine,cytokine, etc.) or other moiety.

In some embodiments, the present invention provides a therapeutic agentconjugated with a trigger agent that is sensitive to (e.g., is cleavedby) hypoxia (e.g., indolequinone). Hypoxia is a feature of severaldisease states, including cancer, inflammation and rheumatoid arthritis,as well as an indicator of respiratory depression (e.g., resulting fromanalgesic drugs).

Advances in the chemistry of bioreductive drug activation have led tothe design of various hypoxia-selective drug delivery systems in whichthe pharmacophores of drugs are masked by reductively cleaved groups. Insome embodiments, the trigger agent is utilizes a quinone, N-oxideand/or (hetero)aromatic nitro groups. For example, a quinone present ina conjugate is reduced to phenol under hypoxia conditions, withspontaneous formation of lactone that serves as a driving force for drugrelease. In some embodiments, a heteroaromatic nitro compound present ina conjugate (e.g., a therapeutic agent conjugated (e.g., directly orindirectly) with a trigger agent) is reduced to either an amine or ahydroxylamine, thereby triggering the spontaneous release of atherapeutic agent. In some embodiments, the trigger agent degrades upondetection of reduced pO2 concentrations (e.g., through use of a redoxlinker).

The concept of pro-drug systems in which the pharmacophores of drugs aremasked by reductively cleavable groups has been widely explored by manyresearch groups and pharmaceutical companies (see, e.g., Beall, H. D.,et al., Journal of Medicinal Chemistry, 1998. 41(24): p. 4755-4766;Ferrer, S., D. P. Naughton, and M. D. Threadgill, Tetrahedron, 2003.59(19): p. 3445-3454; Naylor, M. A., et al., Journal of MedicinalChemistry, 1997. 40(15): p. 2335-2346; Phillips, R. M., et al., Journalof Medicinal Chemistry, 1999. 42(20): p. 4071-4080; Zhang, Z., et al.,Organic & Biomolecular Chemistry, 2005. 3(10): p. 1905-1910; each ofwhich are herein incorporated by reference in their entireties). Severalsuch hypoxia activated pro-drugs have been advanced to clinicalinvestigations, and work in relevant oxygen concentrations to preventcerebral damage. The present invention is not limited to particularhypoxia activated trigger agents. In some embodiments, the hypoxiaactivated trigger agents include, but are not limited to,indolequinones, nitroimidazoles, and nitroheterocycles (see, e.g.,Damen, E. W. P., et al., Bioorganic & Medicinal Chemistry, 2002. 10(1):p. 71-77; Hay, M. P., et al., Journal of Medicinal Chemistry, 2003.46(25): p. 5533-5545; Hay, M. P., et al., Journal of the ChemicalSociety-Perkin Transactions 1, 1999(19): p. 2759-2770; each hereinincorporated by reference in their entireties).

In some embodiments, the trigger agent is sensitive to (e.g., is cleavedby) and/or associates with a tumor-associated enzyme. For example, insome embodiments, the trigger agent that is sensitive to (e.g., iscleaved by) and/or associates with a glucuronidase. Glucuronic acid canbe attached to several anticancer drugs via various linkers. Theseanticancer drugs include, but are not limited to, doxorubicin,paclitaxel, docetaxel, 5-fluorouracil, 9-aminocamtothecin, as well asother drugs under development. These pro-drugs are generally stable atphysiological pH and are significantly less toxic than the parent drugs.

In some embodiments, the trigger agent is sensitive to (e.g., is cleavedby) and/or associates with brain enzymes. For example, trigger agentssuch as indolequinone are reduced by brain enzymes such as, for example,diaphorase (DT-diaphorase) (see, e.g., Damen, E. W. P., et al.,Bioorganic & Medicinal Chemistry, 2002. 10(1): p. 71-77; hereinincorporated by reference in its entirety). For example, in suchembodiments, the antagonist is only active when released during hypoxiato prevent respiratory failure.

In some embodiments, the trigger agent is sensitive to (e.g., is cleavedby) and/or associates with a protease. The present invention is notlimited to any particular protease. In some embodiments, the protease isa cathepsin. In some embodiments, a trigger comprises a Lys-Phe-PABCmoiety (e.g., that acts as a trigger). In some embodiments, aLys-Phe-PABC moiety linked to doxorubicin, mitomycin C, and paclitaxelare utilized as a trigger-therapeutic conjugate in a dendrimer conjugateprovided herein (e.g., that serve as substrates for lysosomal cathepsinB or other proteases expressed (e.g., overexpressed) in tumor cells. Insome embodiments, utilization of a 1,6-elimination spacer/linker isutilized (e.g., to permit release of therapeutic drug post activation oftrigger).

In some embodiments, the trigger agent is sensitive to (e.g., is cleavedby) and/or associates with plasmin. The serine protease plasmin is overexpressed in many human tumor tissues. Tripeptide specifiers (e.g.,including, but not limited to, Val-Leu-Lys) have been identified andlinked to anticancer drugs through elimination or cyclization linkers.

In some embodiments, the trigger agent is sensitive to (e.g., is cleavedby) and/or associates with a matrix metalloprotease (MMP). In someembodiments, the trigger agent is sensitive to (e.g., is cleaved by)and/or that associates with β-Lactamase (e.g., a β-Lactamase activatedcephalosporin-based pro-drug).

In some embodiments, the trigger agent is sensitive to (e.g., is cleavedby) and/or activated by a receptor (e.g., expressed on a target cell(e.g., a tumor cell)).

In some embodiments, the trigger agent is sensitive to (e.g., is cleavedby) and/or activated by a nucleic acid. Nucleic acid triggered catalyticdrug release can be utilized in the design of chemotherapeutic agents.Thus, in some embodiments, disease specific nucleic acid sequence isutilized as a drug releasing enzyme-like catalyst (e.g., via complexformation with a complimentary catalyst-bearing nucleic acid and/oranalog). In some embodiments, the release of a therapeutic agent isfacilitated by the therapeutic component being attached to a labileprotecting group, such as, for example, cisplatin or methotrexate beingattached to a photolabile protecting group that becomes released bylaser light directed at cells emitting a color of fluorescence (e.g., inaddition to and/or in place of target activated activation of a triggercomponent of a dendrimer conjugate). In some embodiments, thetherapeutic device also may have a component to monitor the response ofthe tumor to therapy. For example, where a therapeutic agent of thedendrimer induces apoptosis of a target cell (e.g., a cancer cell (e.g.,a prostate cancer cell)), the caspase activity of the cells may be usedto activate a green fluorescence. This allows apoptotic cells to turnorange, (combination of red and green) while residual cells remain red.Any normal cells that are induced to undergo apoptosis in collateraldamage fluoresce green.

In some embodiments, a dendrimer is conjugated (e.g., directly orindirectly (e.g., via a triazine compound)) with a targeting agent. Thepresent invention is not limited to any particular targeting agent. Insome embodiments, targeting agents are conjugated to a dendrimer (eg.,directly or indirectly) for delivery to desired body regions (e.g., tothe central nervous system (CNS); to a tumor). The targeting agents arenot limited to targeting specific body regions.

In some embodiments, the targeting agent is a moiety that has affinityfor a tumor associated factor. For example, a number of targeting agentsare contemplated to be useful in the present invention including, butnot limited to, RGD sequences, low-density lipoprotein sequences, aNAALADase inhibitor, epidermal growth factor, and other agents that bindwith specificity to a target cell (e.g., a cancer cell)).

The present invention is not limited to cancer and/or tumor targetingagents. Indeed, multifunctional dendrimers can be targeted (e.g., via alinker conjugated to the dendrimer wherein the linker comprises atargeting agent) to a variety of target cells or tissues (e.g., to abiologically relevant environment) via conjugation to an appropriatetargeting agent. For example, in some embodiments, the targeting agentis a moiety that has affinity for an inflammatory factor (e.g., acytokine or a cytokine receptor moiety (e.g., TNF-α receptor)). In someembodiments, the targeting agent is a sugar, peptide, antibody orantibody fragment, hormone, hormone receptor, or the like.

In some embodiments of the present invention, the targeting agentincludes, but is not limited to an antibody, receptor ligand, hormone,vitamin, and antigen, however, the present invention is not limited bythe nature of the targeting agent. In some embodiments, the antibody isspecific for a disease-specific antigen. In some embodiments, thedisease-specific antigen comprises a tumor-specific antigen. In someembodiments, the receptor ligand includes, but is not limited to, aligand for CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2receptor, glycoprotein, and VEGFR. In some embodiments, the receptorligand is folic acid.

Antibodies can be generated to allow for the targeting of antigens orimmunogens (e.g., tumor, tissue or pathogen specific antigens) onvarious biological targets (e.g., pathogens, tumor cells, normaltissue). Such antibodies include, but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments, and an Fab expressionlibrary.

In some embodiments, the targeting agent is an antibody. In someembodiments, the antibodies recognize, for example, tumor-specificepitopes (e.g., TAG-72 (See, e.g., Kjeldsen et al., Cancer Res.48:2214-2220 (1988); U.S. Pat. Nos. 5,892,020; 5,892,019; and 5,512,443;each herein incorporated by reference in their entireties); humancarcinoma antigen (See, e.g., U.S. Pat. Nos. 5,693,763; 5,545,530; and5,808,005; each herein incorporated by reference in their entireties);TP1 and TP3 antigens from osteocarcinoma cells (See, e.g., U.S. Pat. No.5,855,866; herein incorporated by reference in its entirety);Thomsen-Friedenreich (TF) antigen from adenocarcinoma cells (See, e.g.,U.S. Pat. No. 5,110,911; herein incorporated by reference in itsentirety); “KC-4 antigen” from human prostrate adenocarcinoma (See,e.g., U.S. Pat. Nos. 4,708,930 and 4,743,543; each herein incorporatedby reference in their entireties); a human colorectal cancer antigen(See, e.g., U.S. Pat. No. 4,921,789; herein incorporated by reference inits entirety); CA125 antigen from cystadenocarcinoma (See, e.g., U.S.Pat. No. 4,921,790; herein incorporated by reference in its entirety);DF3 antigen from human breast carcinoma (See, e.g., U.S. Pat. Nos.4,963,484 and 5,053,489; each herein incorporated by reference in theirentireties); a human breast tumor antigen (See, e.g., U.S. Pat. No.4,939,240: herein incorporated by reference in its entirety); p97antigen of human melanoma (See, e.g., U.S. Pat. No. 4,918,164: hereinincorporated by reference in its entirety); carcinoma ororosomucoid-related antigen (CORA) (See, e.g., U.S. Pat. No. 4,914,021;herein incorporated by reference in its entirety); a human pulmonarycarcinoma antigen that reacts with human squamous cell lung carcinomabut not with human small cell lung carcinoma (See, e.g., U.S. Pat. No.4,892,935; herein incorporated by reference in its entirety); T and Tnhaptens in glycoproteins of human breast carcinoma (See, e.g., Springeret al., Carbohydr. Res. 178:271-292 (1988); herein incorporated byreference in its entirety), MSA breast carcinoma glycoprotein termed(See, e.g., Tjandra et al., Br. J. Surg. 75:811-817 (1988); hereinincorporated by reference in its entirety); MFGM breast carcinomaantigen (See, e.g., Ishida et al., Tumor Biol. 10:12-22 (1989); hereinincorporated by reference in its entirety); DU-PAN-2 pancreaticcarcinoma antigen (See, e.g., Lan et al., Cancer Res. 45:305-310 (1985);herein incorporated by reference in its entirety); CA125 ovariancarcinoma antigen (See, e.g., Hanisch et al., Carbohydr. Res. 178:29-47(1988); herein incorporated by reference in its entirety); YH206 lungcarcinoma antigen (See, e.g., Hinoda et al., (1988) Cancer J. 42:653-658(1988); herein incorporated by reference in its entirety).

In some embodiments, the targeting agents target the central nervoussystem (CNS). In some embodiments, where the targeting agent is specificfor the CNS, the targeting agent is transferrin (see, e.g., Daniels, T.R., et al., Clinical Immunology, 2006. 121(2): p. 159-176; Daniels, T.R., et al., Clinical Immunology, 2006. 121(2): p. 144-158; each hereinincorporated by reference in their entireties). Transferrin has beenutilized as a targeting vector to transport, for example, drugs,liposomes and proteins across the blood-brain barrier (BBB) by receptormediated transcytosis (see, e.g., Smith, M. W. and M. Gumbleton, Journalof Drug Targeting, 2006. 14(4): p. 191-214; herein incorporated byreference in its entirety). In some embodiments, the targeting agentstarget neurons within the central nervous system (CNS). In someembodiments, where the targeting agent is specific for neurons withinthe CNS, the targeting agent is a synthetic tetanus toxin fragment(e.g., a 12 amino acid peptide (Tet 1) (HLNILSTLWKYR) (SEQ ID NO: 2))(see, e.g., Liu, J. K., et al., Neurobiology of Disease, 2005. 19(3): p.407-418; herein incorporated by reference in its entirety).

In some embodiments, a dendrimer is conjugated (e.g., directly orindirectly (e.g., via a triazine compound)) to an imaging agent. Amultiplicity of imaging agents find use in the present invention. Insome embodiments, a multifunctional dendrimer comprises at least oneimaging agent that can be readily imaged. The present invention is notlimited by the nature of the imaging component used. In some embodimentsof the present invention, imaging modules comprise surface modificationsof quantum dots (See e.g., Chan and Nie, Science 281:2016 (1998)) suchas zinc sulfide-capped cadmium selenide coupled to biomolecules(Sooklal, Adv. Mater., 10:1083 (1998)).

In some embodiments, once a component(s) of a targeted multifunctionaldendrimer has attached to (or been internalized into) a target cell(e.g., tumor cell and or inflammatory cell), one or more modules onserves to image its location. In some embodiments, chelated paramagneticions, such as Gd(III)-diethylenetriaminepentaacetic acid (Gd(III)-DTPA),are conjugated to the multifunctional dendrimer. Other paramagnetic ionsthat may be useful in this context include, but are not limited to,gadolinium, manganese, copper, chromium, iron, cobalt, erbium, nickel,europium, technetium, indium, samarium, dysprosium, ruthenium,ytterbium, yttrium, and holmium ions and combinations thereof.

Dendrimeric gadolinium contrast agents have even been used todifferentiate between benign and malignant breast tumors using dynamicMRI, based on how the vasculature for the latter type of tumor imagesmore densely (Adam et al., Ivest. Rad. 31:26 (1996)). Thus, MRI providesa particularly useful imaging system of the present invention.

Multifunctional dendrimers allow functional microscopic imaging oftumors and provide improved methods for imaging. The methods find use invivo, in vitro, and ex vivo. For example, in one embodiment, dendrimerfunctional groups are designed to emit light or other detectable signalsupon exposure to light. Although the labeled functional groups may bephysically smaller than the optical resolution limit of the microscopytechnique, they become self-luminous objects when excited and arereadily observable and measurable using optical techniques. In someembodiments of the present invention, sensing fluorescent biosensors ina microscope involves the use of tunable excitation and emission filtersand multiwavelength sources (See, e.g., Farkas et al., SPEI 2678:200(1997); herein incorporated by reference in its entirety). Inembodiments where the imaging agents are present in deeper tissue,longer wavelengths in the Near-infrared (NMR) are used (See e.g., Lesteret al., Cell Mol. Biol. 44:29 (1998); herein incorporated by referencein its entirety). Biosensors that find use with the present inventioninclude, but are not limited to, fluorescent dyes and molecular beacons.

In some embodiments of the present invention, in vivo imaging isaccomplished using functional imaging techniques. Functional imaging isa complementary and potentially more powerful techniques as compared tostatic structural imaging. Functional imaging is best known for itsapplication at the macroscopic scale, with examples including functionalMagnetic Resonance Imaging (fMRI) and Positron Emission Tomography(PET). However, functional microscopic imaging may also be conducted andfind use in in vivo and ex vivo analysis of living tissue. Functionalmicroscopic imaging is an efficient combination of 3-D imaging, 3-Dspatial multispectral volumetric assignment, and temporal sampling: inshort a type of 3-D spectral microscopic movie loop. Interestingly,cells and tissues autofluoresce when excited by several wavelengths,providing much of the basic 3-D structure needed to characterize severalcellular components (e.g., the nucleus) without specific labeling.Oblique light illumination is also useful to collect structuralinformation and is used routinely. As opposed to structural spectralmicroimaging, functional spectral microimaging may be used withbiosensors, which act to localize physiologic signals within the cell ortissue. For example, in some embodiments, biosensor-comprising pro-drugcomplexes are used to image upregulated receptor families such as thefolate or EGF classes. In such embodiments, functional biosensingtherefore involves the detection of physiological abnormalities relevantto carcinogenesis or malignancy, even at early stages. A number ofphysiological conditions may be imaged using the compositions andmethods of the present invention including, but not limited to,detection of nanoscopic biosensors for pH, oxygen concentration, Ca²+concentration, and other physiologically relevant analytes.

In some embodiments, the present invention provides multifunctionaldendrimers (e.g., conjugated with a triazine compound) having abiological monitoring component. The biological monitoring or sensingcomponent of a multifunctional dendrimer is one that can monitor theparticular response in a target cell (e.g., tumor cell) induced by anagent (e.g., a therapeutic agent provided by a multifunctionaldendrimer). While the present invention is not limited to any particularmonitoring system, the invention is illustrated by methods andcompositions for monitoring cancer treatments. In preferred embodimentsof the present invention, the agent induces apoptosis in cells andmonitoring involves the detection of apoptosis. In some embodiments, themonitoring component is an agent that fluoresces at a particularwavelength when apoptosis occurs. For example, in a preferredembodiment, caspase activity activates green fluorescence in themonitoring component. Apoptotic cancer cells, which have turned red as aresult of being targeted by a particular signature with a red label,turn orange while residual cancer cells remain red. Normal cells inducedto undergo apoptosis (e.g., through collateral damage), if present, willfluoresce green.

In these embodiments, fluorescent groups such as fluorescein areemployed in the imaging agent. Fluorescein is easily attached to thedendrimer surface via the isothiocyanate derivatives, available fromMOLECULAR PROBES, Inc. This allows the multifunctional dendrimer orcomponents thereof to be imaged with the cells via confocal microscopy.Sensing of the effectiveness of the multifunctional dendrimer orcomponents thereof is preferably achieved by using fluorogenic peptideenzyme substrates. For example, apoptosis caused by the therapeuticagent results in the production of the peptidase caspase-1 (ICE).CALBIOCHEM sells a number of peptide substrates for this enzyme thatrelease a fluorescent moiety. A particularly useful peptide for use inthe present invention is: MCA-Tyr-Glu-Val-Asp-Gly-Trp-Lys-(DNP)-NH₂ (SEQID NO: 1) where MCA is the (7-methoxycoumarin-4-yl)acetyl and DNP is the2,4-dinitrophenyl group (See, e.g., Talanian et al., J. Biol. Chem.,272: 9677 (1997); herein incorporated by reference in its entirety). Inthis peptide, the MCA group has greatly attenuated fluorescence, due tofluorogenic resonance energy transfer (FRET) to the DNP group. When theenzyme cleaves the peptide between the aspartic acid and glycineresidues, the MCA and DNP are separated, and the MCA group stronglyfluoresces green (excitation maximum at 325 nm and emission maximum at392 nm). In some embodiments, the lysine end of the peptide is linked topro-drug complex, so that the MCA group is released into the cytosolwhen it is cleaved. The lysine end of the peptide is a useful synthetichandle for conjugation because, for example, it can react with theactivated ester group of a bifunctional linker such as Mal-PEG-OSu. Thusthe appearance of green fluorescence in the target cells produced usingthese methods provides a clear indication that apoptosis has begun (ifthe cell already has a red color from the presence of aggregated quantumdots, the cell turns orange from the combined colors).

Additional fluorescent dyes that find use with the present inventioninclude, but are not limited to, acridine orange, reported as sensitiveto DNA changes in apoptotic cells (see, e.g., Abrams et al., Development117:29 (1993); herein incorporated by reference in its entirety) andcis-parinaric acid, sensitive to the lipid peroxidation that accompaniesapoptosis (see, e.g., Hockenbery et al., Cell 75:241 (1993); hereinincorporated by reference in its entirety). It should be noted that thepeptide and the fluorescent dyes are merely exemplary. It iscontemplated that any peptide that effectively acts as a substrate for acaspase produced as a result of apoptosis finds use with the presentinvention.

In some embodiments, conjugation between a dendrimer (e.g., terminal armof a dendrimer) and a functional group or between functional groups isaccomplished through use of a 1,3-dipolar cycloaddition reaction (“clickchemistry”). Moreover, in some embodiments, conjugation between adendrimer (e.g., terminal arm of a dendrimer) and a triazine compound(e.g.,

is accomplished through use of a 1,3-dipolar cycloaddition reaction(“click chemistry”). ‘Click chemistry’ involves, for example, thecoupling of two different moieties (e.g., a therapeutic agent and afunctional group) (e.g., a first functional group and a secondfunctional group) via a 1,3-dipolar cycloaddition reaction between analkyne moiety (or equivalent thereof) on the surface of the first moeityand an azide moiety (e.g., present on a triazine composition of thepresent invention) (or equivalent thereof) (or any active end group suchas, for example, a primary amine end group, a hydroxyl end group, acarboxylic acid end group, a thiol end group, etc.) on the secondmoiety. ‘Click chemistry’ is an attractive coupling method because, forexample, it can be performed with a wide variety of solvent conditionsincluding aqueous environments. For example, the stable triazole ringthat results from coupling the alkyne with the azide is frequentlyachieved at quantitative yields and is considered to be biologicallyinert (see, e.g., Rostovtsev, V. V.; et al., AngewandteChemie-International Edition 2002, 41, (14), 2596; Wu, P.; et al.,Angewandte Chemie-International Edition 2004, 43, (30), 3928-3932; eachherein incorporated by reference in their entireties).

In some embodiments, conjugation between a dendrimer (e.g., a terminalarm of a dendrimer) and a functional ligand is accomplished during a“one-pot” reaction. The term “one-pot synthesis reaction” or equivalentsthereof, e.g., “1-pot”, “one pot”, etc., refers to a chemical synthesismethod in which all reactants are present in a single vessel. Reactantsmay be added simultaneously or sequentially, with no limitation as tothe duration of time elapsing between introduction of sequentially addedreactants. In some embodiments, a one-pot reaction occurs wherein ahydroxyl-terminated dendrimer (e.g., HO-PAMAM dendrimer) is reacted withone or more functional ligands (e.g., a therapeutic agent, a pro-drug, atrigger agent, a targeting agent, an imaging agent) in one vessel, suchconjugation being facilitated by ester coupling agents (e.g.,2-chloro-1-methylpyridinium iodide and 4-(dimethylamino) pyridine) (see,e.g., International Patent Application No. PCT/US2010/042556, hereinincorporated by reference in its entirety).

Functionalized nanoparticles (e.g., dendrimers) often contain moieties(including but not limited to ligands, functional ligands, conjugates,therapeutic agents, targeting agents, imaging agents, fluorophores) thatare conjugated to the periphery. Such moieties may for example beconjugated to one or more dendrimer branch termini. Classical multi-stepconjugation strategies used during the synthesis of functionalizeddendrimers generate a stochastic distribution of products with differingnumbers of ligands attached per dendrimer molecule, thereby creating apopulation of dendrimers with a wide distribution in the numbers ofligands attached. The low structural uniformity of such dendrimerpopulations negatively affects properties such as therapeutic potency,pharmacokinetics, or effectiveness for multivalent targeting.Difficulties in quantifying and resolving such populations to yieldsamples with sufficient structural uniformity can pose challenges.However, in some embodiments, use of separation methods (e.g., reversephase chromatography) customized for optimal separation of dendrimerpopulations in conjunction with peak fitting analysis methods allowsisolation and identification of subpopulations of functionalizeddendrimers with high structural uniformity (see, e.g., U.S. ProvisionalPat. App. No. 61/237,172; herein incorporated by reference in itsentirety). In certain embodiments, such methods and systems provide adendrimer product made by the process comprising: a) conjugation of atleast one ligand type to a dendrimer to yield a population ofligand-conjugated dendrimers; b) separation of the population ofligand-conjugated dendrimers with reverse phase HPLC to result insubpopulations of ligand-conjugated dendrimers indicated by achromatographic trace; and c) application of peak fitting analysis tothe chromatographic trace to identify subpopulations ofligand-conjugated dendrimers wherein the structural uniformity of ligandconjugates per molecule of dendrimer within said subpopulation is, e.g.,approximately 80% or more.

The present invention is not limited by the type of therapeutic agentdelivered via multifunctional dendrimers of the present invention. Forexample, a therapeutic agent may be any agent selected from the groupcomprising, but not limited to, a pain relief agent, a pain relief agentantagonist, a chemotherapeutic agent, an anti-oncogenic agent, ananti-angiogenic agent, a tumor suppressor agent, an anti-microbialagent, or an expression construct comprising a nucleic acid encoding atherapeutic protein.

Indeed, in some embodiments of the present invention, methods andcompositions are provided for the treatment of inflammatory diseases(e.g., dendrimers conjugated with therapeutic agents configured fortreating inflammatory diseases). Inflammatory diseases include but arenot limited to arthritis, rheumatoid arthritis, psoriatic arthritis,osteoarthritis, degenerative arthritis, polymyalgia rheumatic,ankylosing spondylitis, reactive arthritis, gout, pseudogout,inflammatory joint disease, systemic lupus erythematosus, polymyositis,and fibromyalgia. Additional types of arthritis include achillestendinitis, achondroplasia, acromegalic arthropathy, adhesivecapsulitis, adult onset Still's disease, anserine bursitis, avascularnecrosis, Behcet's syndrome, bicipital tendinitis, Blount's disease,brucellar spondylitis, bursitis, calcaneal bursitis, calciumpyrophosphate dihydrate deposition disease (CPPD), crystal depositiondisease, Caplan's syndrome, carpal tunnel syndrome, chondrocalcinosis,chondromalacia patellae, chronic synovitis, chronic recurrent multifocalosteomyelitis, Churg-Strauss syndrome, Cogan's syndrome,corticosteroid-induced osteoporosis, costosternal syndrome, CRESTsyndrome, cryoglobulinemia, degenerative joint disease, dermatomyositis,diabetic finger sclerosis, diffuse idiopathic skeletal hyperostosis(DISH), discitis, discoid lupus erythematosus, drug-induced lupus,Duchenne's muscular dystrophy, Dupuytren's contracture, Ehlers-Danlossyndrome, enteropathic arthritis, epicondylitis, erosive inflammatoryosteoarthritis, exercise-induced compartment syndrome, Fabry's disease,familial Mediterranean fever, Farber's lipogranulomatosis, Felty'ssyndrome, Fifth's disease, flat feet, foreign body synovitis, Freiberg'sdisease, fungal arthritis, Gaucher's disease, giant cell arteritis,gonococcal arthritis, Goodpasture's syndrome, granulomatous arteritis,hemarthrosis, hemochromatosis, Henoch-Schonlein purpura, Hepatitis Bsurface antigen disease, hip dysplasia, Hurler syndrome, hypermobilitysyndrome, hypersensitivity vasculitis, hypertrophic osteoarthropathy,immune complex disease, impingement syndrome, Jaccoud's arthropathy,juvenile ankylosing spondylitis, juvenile dermatomyositis, juvenilerheumatoid arthritis, Kawasaki disease, Kienbock's disease,Legg-Calve-Perthes disease, Lesch-Nyhan syndrome, linear scleroderma,lipoid dermatoarthritis, Lofgren's syndrome, Lyme disease, malignantsynovioma, Marfan's syndrome, medial plica syndrome, metastaticcarcinomatous arthritis, mixed connective tissue disease (MCTD), mixedcryoglobulinemia, mucopolysaccharidosis, multicentricreticulohistiocytosis, multiple epiphyseal dysplasia, mycoplasmalarthritis, myofascial pain syndrome, neonatal lupus, neuropathicarthropathy, nodular panniculitis, ochronosis, olecranon bursitis,Osgood-Schlatter's disease, osteoarthritis, osteochondromatosis,osteogenesis imperfecta, osteomalacia, osteomyelitis, osteonecrosis,osteoporosis, overlap syndrome, pachydermoperiostosis Paget's disease ofbone, palindromic rheumatism, patellofemoral pain syndrome,Pellegrini-Stieda syndrome, pigmented villonodular synovitis, piriformissyndrome, plantar fasciitis, polyarteritis nodos, Polymyalgia rheumatic,polymyositis, popliteal cysts, posterior tibial tendinitis, Pott'sdisease, prepatellar bursitis, prosthetic joint infection,pseudoxanthoma elasticum, psoriatic arthritis, Raynaud's phenomenon,reactive arthritis/Reiter's syndrome, reflex sympathetic dystrophysyndrome, relapsing polychondritis, retrocalcaneal bursitis, rheumaticfever, rheumatoid vasculitis, rotator cuff tendinitis, sacroiliitis,salmonella osteomyelitis, sarcoidosis, saturnine gout, Scheuermann'sosteochondritis, scleroderma, septic arthritis, seronegative arthritis,shigella arthritis, shoulder-hand syndrome, sickle cell arthropathy,Sjogren's syndrome, slipped capital femoral epiphysis, spinal stenosis,spondylolysis, staphylococcus arthritis, Stickler syndrome, subacutecutaneous lupus, Sweet's syndrome, Sydenham's chorea, syphiliticarthritis, systemic lupus erythematosus (SLE), Takayasu's arteritis,tarsal tunnel syndrome, tennis elbow, Tietse's syndrome, transientosteoporosis, traumatic arthritis, trochanteric bursitis, tuberculosisarthritis, arthritis of Ulcerative colitis, undifferentiated connectivetissue syndrome (UCTS), urticarial vasculitis, viral arthritis,Wegener's granulomatosis, Whipple's disease, Wilson's disease, andyersinial arthritis.

In some embodiments, the conjugated dendrimers of the present inventionconfigured for treating autoimmune disorders and/or inflammatorydisorders (e.g., rheumatoid arthritis) are co-administered to a subject(e.g., a human suffering from an autoimmune disorder and/or aninflammatory disorder) with a therapeutic agent configured for treatingautoimmune disorders and/or inflammatory disorders (e.g., rheumatoidarthritis). Examples of such agents include, but are not limited to,disease-modifying antirheumatic drugs (e.g., leflunomide, methotrexate,sulfasalazine, hydroxychloroquine), biologic agents (e.g., rituximab,infliximab, etanercept, adalimumab, golimumab), nonsteroidalanti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen,naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen,tramadol), immunomodulators (e.g., anakinra, abatacept), andglucocorticoids (e.g., prednisone, methylprednisone), IL-1 inhibitors,and metalloprotease inhibitors.

In some embodiments, the medical condition and/or disease is pain (e.g.,chronic pain, mild pain, recurring pain, severe pain, etc.). In someembodiments, the conjugated dendrimers of the present invention areconfigured to deliver pain relief agents to a subject (see, e.g., U.S.patent application Ser. No. 12/570,977; herein incorporated by referencein its entirety). In some embodiments, the dendrimer conjugates areconfigured to deliver pain relief agents and pain relief agentantagonists to counter the side effects of pain relief agents (see,e.g., U.S. patent application Ser. No. 12/570,977; herein incorporatedby reference in its entirety). The dendrimer conjugates are not limitedto treating a particular type of pain and/or pain resulting from adisease (see, e.g., U.S. patent application Ser. No. 12/570,977; hereinincorporated by reference in its entirety). Examples include, but arenot limited to, pain resulting from trauma (e.g., trauma experienced ona battlefield, trauma experienced in an accident (e.g., car accident)).In some embodiments, the dendrimer conjugates of the present inventionare configured such that they are readily cleared from the subject(e.g., so that there is little to no detectable toxicity at efficaciousdoses) (see, e.g., U.S. patent application Ser. No. 12/570,977; hereinincorporated by reference in its entirety).

In some embodiments, the disease is cancer. The present invention is notlimited by the type of cancer treated using the compositions and methodsof the present invention. Indeed, a variety of cancer can be treatedincluding, but not limited to, prostate cancer, colon cancer, breastcancer, lung cancer and epithelial cancer.

In some embodiments, the disease is a neoplastic disease, selected from,but not limited to, leukemia, acute leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, myeloblastic, promyelocytic,myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronicmyelocytic, (granulocytic) leukemia, chronic lymphocytic leukemia,Polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease,Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease,solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, and neuroblastomaretinoblastoma. In some embodiments, thedisease is an inflammatory disease selected from the group consistingof, but not limited to, eczema, inflammatory bowel disease, rheumatoidarthritis, asthma, psoriasis, ischemia/reperfusion injury, ulcerativecolitis and acute respiratory distress syndrome. In some embodiments,the disease is a viral disease selected from the group consisting of,but not limited to, viral disease caused by hepatitis B, hepatitis C,rotavirus, human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), human T-cell lymphotropic virustype I (HTLV-I), human T-cell lymphotropic virus type II (HTLV-II),AIDS, DNA viruses such as hepatitis type B and hepatitis type C virus;parvoviruses, such as adeno-associated virus and cytomegalovirus;papovaviruses such as papilloma virus, polyoma viruses, and SV40;adenoviruses; herpes viruses such as herpes simplex type I (HSV-I),herpes simplex type II (HSV-II), and Epstein-Barr virus; poxviruses,such as variola (smallpox) and vaccinia virus; and RNA viruses, such ashuman immunodeficiency virus type I (HIV-I), human immunodeficiencyvirus type II (HIV-II), human T-cell lymphotropic virus type I (HTLV-I),human T-cell lymphotropic virus type II (HTLV-II), influenza virus,measles virus, rabies virus, Sendai virus, picornaviruses such aspoliomyelitis virus, coxsackieviruses, rhinoviruses, reoviruses,togaviruses such as rubella virus (German measles) and Semliki forestvirus, arboviruses, and hepatitis type A virus.

In some embodiments, the dendrimer is conjugated with one or moreanti-cancer agents. In some embodiments, the dendrimer isco-administered with one or more anti-cancer agents. Examples ofanti-cancer agents include, but are not limited to, Acivicin;Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin;Aldesleukin; Alitretinoin; Allopurinol Sodium; Altretamine; Ambomycin;Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole;Annonaceous Acetogenins; Anthramycin; Asimicin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bexarotene;Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Bullatacin; Busulfan;Cabergoline; Cactinomycin; Calusterone; Caracemide; Carbetimer;Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin;Cedefingol; Celecoxib; Chlorambucil; Cirolemycin; Cisplatin; Cladribine;Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; DACA(N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin;Daunorubicin Hydrochloride; Daunomycin; Decitabine; Denileukin Diftitox;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized OilI 131; Etoposide; Etoposide Phosphate; Etoprine; FadrozoleHydrochloride; Fazarabine; Fenretinide; Floxuridine; FludarabinePhosphate; Fluorouracil; 5-FdUMP; Fluorocitabine; Fosquidone; FostriecinSodium; FK-317; FK-973; FR-66979; FR-900482; Gemcitabine; GeimcitabineHydrochloride; Gemtuzumab Ozogamicin; Gold Au 198; Goserelin Acetate;Guanacone; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-1a; Interferon Gamma-1b; Iproplatin;Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; LeuprolideAcetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine;Losoxantrone Hydrochloride; Masoprocol; Maytansine; MechlorethamineHydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan;Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Methoxsalen; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin;Mitogillin; Mitomalcin; Mitomycin; Mytomycin C; Mitosper; Mitotane;Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;Oprelvekin; Ormaplatin; Oxisuran; Paclitaxel; Pamidronate Disodium;Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rituximab; Rogletimide; Rolliniastatin;Safingol; Safingol Hydrochloride; Samarium/Lexidronam; Semustine;Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Squamocin; Squamotacin;Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur;Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine;Tomudex; TOP-53; Topotecan Hydrochloride; Toremifene Citrate;Trastuzumab; Trestolone Acetate; Triciribine Phosphate; Trimetrexate;Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; UracilMustard; Uredepa; Valrubicin; Vapreotide; Verteporfin; Vinblastine;Vinblastine Sulfate; Vincristine; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride; 2-Chlorodeoxyadenosine; 2′-Deoxyformycin;9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid;2-chloro-2′-arabino-fluoro-2′-deoxyadenosine;2-chloro-2′-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R;CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlorethamine);cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan;N-methyl-N-nitrosourea (MNU); N,N′-Bis(2-chloroethyl)-N-nitrosourea(BCNU); N-(2-chloroethyl)-N′-cyclohex-yl-N-nitrosourea (CCNU);N-(2-chloroethyl)-N′-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU);N-(2-chloroethyl)-N′-(diethyl)ethylphosphonate-N-nit-rosourea(fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide;temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin;Carboplatin; Ormaplatin; Oxaliplatin; C1-973; DWA 2114R; JM216; JM335;Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine;6-Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide; 9-aminocamptothecin; Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin;darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D);amsacrine; pyrazoloacridine; all-trans retinol;14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-Hydroxyphenyl)retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid;fludarabine (2-F-ara-AMP); and 2-chlorodeoxyadenosine (2-Cda). Otheranti-cancer agents include, but are not limited to, Antiproliferativeagents (e.g., Piritrexim Isothionate), Antiprostatic hypertrophy agent(e.g., Sitogluside), Benign prostatic hyperplasia therapy agents (e.g.,Tamsulosin Hydrochloride), Prostate growth inhibitor agents (e.g.,Pentomone), and Radioactive agents: Fibrinogen 1 125; Fludeoxyglucose F18; Fluorodopa F 18; Insulin I 125; Insulin I 131; Iobenguane I 123;Iodipamide Sodium I 131; Iodoantipyrine I 131; Iodocholesterol I 131;Iodohippurate Sodium I 123; Iodohippurate Sodium I 125; IodohippurateSodium I 131; Iodopyracet I 125; Iodopyracet I 131; IofetamineHydrochloride I 123; Iomethin I 125; Iomethin I 131; Iothalamate SodiumI 125; Iothalamate Sodium I 131; Iotyrosine I 131; Liothyronine I 125;Liothyronine I 131; Merisoprol Acetate Hg 197; Merisoprol Acetate Hg203; Merisoprol Hg 197; Selenomethionine Se 75; Technetium Tc 99mAntimony Trisulfide Colloid; Technetium Tc 99m Bicisate; Technetium Tc99m Disofenin; Technetium Tc 99m Etidronate; Technetium Tc 99mExametazime; Technetium Tc 99m Furifosmin; Technetium Tc 99m Gluceptate;Technetium Tc 99m Lidofenin; Technetium Tc 99m Mebrofenin; Technetium Tc99m Medronate; Technetium Tc 99m Medronate Disodium; Technetium Tc 99mMertiatide; Technetium Tc 99m Oxidronate; Technetium Tc 99m Pentetate;Technetium Tc 99m Pentetate Calcium Trisodium; Technetium Tc 99mSestamibi; Technetium Tc 99m Siboroxime; Technetium Tc 99m Succimer;Technetium Tc 99m sulfur Colloid; Technetium Tc 99m Teboroxime;Technetium Tc 99m Tetrofosmin; Technetium Tc 99m Tiatide; Thyroxine I125; Thyroxine I 131; Tolpovidone I 131; Triolein I 125; and Triolein I131.

Additional anti-cancer agents include, but are not limited toanti-cancer Supplementary Potentiating Agents: Tricyclic anti-depressantdrugs (e.g., imipramine, desipramine, amitryptyline, clomipramine,trimipramine, doxepin, nortriptyline, protriptyline, amoxapine andmaprotiline); non-tricyclic anti-depressant drugs (e.g., sertraline,trazodone and citalopram); Ca⁺⁺ antagonists (e.g., verapamil,nifedipine, nitrendipine and caroverine); Calmodulin inhibitors (e.g.,prenylamine, trifluoroperazine and clomipramine); Amphotericin B;Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g.,quinidine); antihypertensive drugs (e.g., reserpine); Thiol depleters(e.g., buthionine and sulfoximine) and Multiple Drug Resistance reducingagents such as Cremaphor EL. Still other anticancer agents include, butare not limited to, annonaceous acetogenins; asimicin; rolliniastatin;guanacone, squamocin, bullatacin; squamotacin; taxanes; paclitaxel;gemcitabine; methotrexate FR-900482; FK-973; FR-66979; FK-317; 5-FU;FUDR; FdUMP; Hydroxyurea; Docetaxel; discodermolide; epothilones;vincristine; vinblastine; vinorelbine; meta-pac; irinotecan; SN-38;10-OH campto; topotecan; etoposide; adriamycin; flavopiridol; Cis-Pt;carbo-Pt; bleomycin; mitomycin C; mithramycin; capecitabine; cytarabine;2-C1-2′ deoxyadenosine; Fludarabine-PO₄; mitoxantrone; mitozolomide;Pentostatin; and Tomudex. One particularly preferred class of anticanceragents are taxanes (e.g., paclitaxel and docetaxel). Another importantcategory of anticancer agent is annonaceous acetogenin.

In some embodiments, the dendrimer is conjugated with one or more painrelief agents. In some embodiments, the dendrimer is co-administeredwith one or more pain relief agents. In some embodiments, the painrelief agents include, but are not limited to, analgesic drugs,anxiolytic drugs, anesthetic drugs, antipsychotic drugs, hypnotic drugs,sedative drugs, and muscle relaxant drugs (see, e.g., U.S. patentapplication Ser. No. 12/570,977; herein incorporated by reference in itsentirety).

In some embodiments, the analgesic drugs include, but are not limitedto, non-steroidal anti-inflammatory drugs, COX-2 inhibitors, andopiates. In some embodiments, the non-steroidal anti-inflammatory drugsare selected from the group consisting of Acetylsalicylic acid(Aspirin), Amoxiprin, Benorylate/Benorilate, Choline magnesiumsalicylate, Diflunisal, Ethenzamide, Faislamine, Methyl salicylate,Magnesium salicylate, Salicyl salicylate, Salicylamide, arylalkanoicacids, Diclofenac, Aceclofenac, Acemethacin, Alclofenac, Bromfenac,Etodolac, Indometacin, Nabumetone, Oxametacin, Proglumetacin, Sulindac,Tolmetin, 2-arylpropionic acids, Ibuprofen, Alminoprofen, Benoxaprofen,Carprofen, Dexibuprofen, Dexketoprofen, Fenbufen, Fenoprofen,Flunoxaprofen, Flurbiprofen, Ibuproxam, Indoprofen, Ketoprofen,Ketorolac, Loxoprofen, Naproxen, Oxaprozin, Pirprofen, Suprofen,Tiaprofenic acid), N-arylanthranilic acids, Mefenamic acid, Flufenamicacid, Meclofenamic acid, Tolfenamic acid, pyrazolidine derivatives,Phenylbutazone, Ampyrone, Azapropazone, Clofezone, Kebuzone, Metamizole,Mofebutazone, Oxyphenbutazone, Phenazone, Sulfinpyrazone, oxicams,Piroxicam, Droxicam, Lornoxicam, Meloxicam, Tenoxicam, sulphonanilides,nimesulide, licofelone, and omega-3 fatty acids. In some embodiments,the COX-2 inhibitors are selected from the group consisting ofCelecoxib, Etoricoxib, Lumiracoxib, Parecoxib, Rofecoxib, andValdecoxib. In some embodiments, the opiate drugs are selected from thegroup consisting of natural opiates, alkaloids, morphine, codeine,thebaine, semi-synthetic opiates, hydromorphone, hydrocodone, oxycodone,oxymorphone, desomorphine, diacetylmorphine (Heroin), nicomorphine,dipropanoylmorphine, diamorphine, benzylmorphine, Buprenorphine,Nalbuphine, Pentazocine, meperidine, diamorphine, ethylmorphine, fullysynthetic opioids, fentanyl, pethidine, Oxycodone, Oxymorphone,methadone, tramadol, Butorphanol, Levorphanol, propoxyphene, endogenousopioid peptides, endorphins, enkephalins, dynorphins, and endomorphins.

In some embodiments, the anxiolytic drugs include, but are not limitedto, benzodiazepines, alprazolam, bromazepam (Lexotan), chlordiazepoxide(Librium), Clobazam, Clonazepam, Clorazepate, Diazepam, Midazolam,Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam, Flunitrazepam,oxazepam (Serax), temazepam (Restoril, Normison, Planum, Tenox, andTemaze, Triazolam, serotonin 1A agonists, Buspirone (BuSpar),barbituates, amobarbital (Amytal), pentobarbital (Nembutal),secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental,Methylphenobarbital, Metharbital, Barbexaclone), hydroxyzine,cannabidiol, valerian, kava (Kava Kava), chamomile, Kratom, Blue Lotusextracts, Sceletium tortuosum (kanna) and bacopa monniera.

In some embodiments, the anesthetic drugs include, but are not limitedto, local anesthetics, procaine, amethocaine, cocaine, lidocaine,prilocalne, bupivacaine, levobupivacaine, ropivacaine, dibucaine,inhaled anesthetics, Desflurane, Enflurane, Halothane, Isoflurane,Nitrous oxide, Sevoflurane, Xenon, intravenous anesthetics,Barbiturates, amobarbital (Amytal), pentobarbital (Nembutal),secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental,Methylphenobarbital, Metharbital, Barbexaclone)), Benzodiazepines,alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium), Clobazam,Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam,temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax),temazepam (Restoril, Normison, Planum, Tenox, and Temaze), Triazolam,Etomidate, Ketamine, and Propofol.

In some embodiments, the antipsychotic drugs include, but are notlimited to, butyrophenones, haloperidol, phenothiazines, Chlorpromazine(Thorazine), Fluphenazine (Prolixin), Perphenazine (Trilafon),Prochlorperazine (Compazine), Thioridazine (Mellaril), Trifluoperazine(Stelazine), Mesoridazine, Promazine, Triflupromazine (Vesprin),Levomepromazine (Nozinan), Promethazine (Phenergan)), thioxanthenes,Chlorprothixene, Flupenthixol (Depixol and Fluanxol), Thiothixene(Navane), Zuclopenthixol (Clopixol & Acuphase)), clozapine, olanzapine,Risperidone (Risperdal), Quetiapine (Seroquel), Ziprasidone (Geodon),Amisulpride (Solian), Paliperidone (Invega), dopamine, bifeprunox,norclozapine (ACP-104), Aripiprazole (Abilify), Tetrabenazine, andCannabidiol.

In some embodiments, the hypnotic drugs include, but are not limited to,Barbiturates, Opioids, benzodiazepines, alprazolam, bromazepam(Lexotan), chlordiazepoxide (Librium), Clobazam, Clonazepam,Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam,nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam(Restoril, Normison, Planum, Tenox, and Temaze), Triazolam,nonbenzodiazepines, Zolpidem, Zaleplon, Zopiclone, Eszopiclone,antihistamines, Diphenhydramine, Doxylamine, Hydroxyzine, Promethazine,gamma-hydroxybutyric acid (Xyrem), Glutethimide, Chloral hydrate,Ethchlorvynol, Levomepromazine, Chlormethiazole, Melatonin, and Alcohol.

In some embodiments, the sedative drugs include, but are not limited to,barbituates, amobarbital (Amytal), pentobarbital (Nembutal),secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental,Methylphenobarbital, Metharbital, Barbexaclone), benzodiazepines,alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium), Clobazam,Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam,temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax),temazepam (Restoril, Normison, Planum, Tenox, and Temaze), Triazolam,herbal sedatives, ashwagandha, catnip, kava (Piper methysticum),mandrake, marijuana, valerian, solvent sedatives, chloral hydrate(Noctec), diethyl ether (Ether), ethyl alcohol (alcoholic beverage),methyl trichloride (Chloroform), nonbenzodiazepine sedatives,eszopiclone (Lunesta), zaleplon (Sonata), zolpidem (Ambien), zopiclone(Imovane, Zimovane)), clomethiazole (clomethiazole),gamma-hydroxybutyrate (GHB), Thalidomide, ethchlorvynol (Placidyl),glutethimide (Doriden), ketamine (Ketalar, Ketaset), methaqualone(Sopor, Quaalude), methyprylon (Noludar), and ramelteon (Rozerem).

In some embodiments, the muscle relaxant drugs include, but are notlimited to, depolarizing muscle relaxants, Succinylcholine, short actingnon-depolarizing muscle relaxants, Mivacurium, Rapacuronium,intermediate acting non-depolarizing muscle relaxants, Atracurium,Cisatracurium, Rocuronium, Vecuronium, long acting non-depolarizingmuscle relaxants, Alcuronium, Doxacurium, Gallamine, Metocurine,Pancuronium, Pipecuronium, and d-Tubocurarine.

In some embodiments, the dendrimer is conjugated with one or more painrelief agent antagonists. In some embodiments, the dendrimer isco-administered with one or more pain relief agent antagonists (see,e.g., U.S. patent application Ser. No. 12/570,977; herein incorporatedby reference in its entirety).

In some embodiments, the pain relief agent antagonists include drugsthat counter the effect of a pain relief agent (e.g., an anestheticantagonist, an analgesic antagonist, a mood stabilizer antagonist, apsycholeptic drug antagonist, a psychoanaleptic drug antagonist, asedative drug antagonist, a muscle relaxant drug antagonist, and ahypnotic drug antagonist). In some embodiments, pain relief agentantagonists include, but are not limited to, a respiratory stimulant,Doxapram, BIMU-8, CX-546, an opiod receptor antagonist, Naloxone,naltrexone, nalorphine, levallorphan, cyprodime, naltrindole,norbinaltorphimine, buprenorphine, a benzodiazepine antagonist,flumazenil, a non-depolarizing muscle relaxant antagonist, andneostigmine.

Where clinical applications are contemplated, in some embodiments of thepresent invention, the dendrimer conjugates are prepared as part of apharmaceutical composition in a form appropriate for the intendedapplication. Generally, this entails preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals. However, in some embodiments of thepresent invention, a straight dendrimer formulation may be administeredusing one or more of the routes described herein.

In preferred embodiments, the dendrimer conjugates are used inconjunction with appropriate salts and buffers to render delivery of thecompositions in a stable manner to allow for uptake by target cells.Buffers also are employed when the dendrimer conjugates are introducedinto a patient. Aqueous compositions comprise an effective amount of thedendrimer conjugates to cells dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions also are referred to asinocula. The phrase “pharmaceutically or pharmacologically acceptable”refers to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. Except insofar as any conventional media or agent is incompatiblewith vectors, cells, or tissues, its use in therapeutic compositions iscontemplated. Supplementary active ingredients may also be incorporatedinto the compositions.

In some embodiments of the present invention, the active compositionsinclude classic pharmaceutical preparations. Administration of thesecompositions according to the present invention is via any common routeso long as the target tissue is available via that route. This includesoral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection.

The active dendrimer conjugates may also be administered parenterally orintraperitoneally or intratumorally. Solutions of the active compoundsas free base or pharmacologically acceptable salts are prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

In some embodiments, a therapeutic agent is released from dendrimerconjugates within a target cell (e.g., within an endosome). This type ofintracellular release (e.g., endosomal disruption of alinker-therapeutic conjugate) is contemplated to provide additionalspecificity for the compositions and methods of the present invention.The present invention provides dendrimers with multiple (e.g., 100-150)reactive sites for the conjugation of linkers and/or functional groupscomprising, but not limited to, therapeutic agents, targeting agents,imaging agents and biological monitoring agents.

The compositions and methods of the present invention are contemplatedto be equally effective whether or not the dendrimer conjugates of thepresent invention comprise a fluorescein (e.g. FITC) imaging agent.Thus, each functional group present in a dendrimer composition is ableto work independently of the other functional groups. Thus, the presentinvention provides dendrimer conjugates that can comprise multiplecombinations of targeting, therapeutic, imaging, and biologicalmonitoring functional groups.

The present invention also provides a very effective and specific methodof delivering molecules (e.g., therapeutic and imaging functionalgroups) to the interior of target cells (e.g., cancer cells). Thus, insome embodiments, the present invention provides methods of therapy thatcomprise or require delivery of molecules into a cell in order tofunction (e.g., delivery of genetic material such as siRNAs).

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. The carrier may be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it may be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, dendrimer conjugates are administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution is suitably buffered, if necessary,and the liquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. For example, one dosage could be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). In some embodiments of the present invention, the activeparticles or agents are formulated within a therapeutic mixture tocomprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose orso. Multiple doses may be administered.

Additional formulations that are suitable for other modes ofadministration include vaginal suppositories and pessaries. A rectalpessary or suppository may also be used. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or the urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1%-2%. Vaginal suppositories or pessaries areusually globular or oviform and weighing about 5 g each. Vaginalmedications are available in a variety of physical forms, e.g., creams,gels or liquids, which depart from the classical concept ofsuppositories. In addition, suppositories may be used in connection withcolon cancer. The dendrimer conjugates also may be formulated asinhalants for the treatment of lung cancer and such like.

In some embodiments, the present invention also provides kits comprisingone or more of the reagents and tools necessary to generate a dendrimerconjugated with one or more triazine compositions (e.g., scaffolds)(e.g., triazine compositions capable of click chemistry for use inone-step synthesis of functionalized dendrimers) (e.g., triazinecompositions having one or more functional groups), and methods of usingsuch dendrimers.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1

Previous experiments involving dendrimer related technologies arelocated in U.S. Pat. Nos. 6,471,968, 7,078,461; U.S. patent applicationSer. Nos. 09/940,243, 10/431,682, 11,503,742, 11,661,465, 11/523,509,12/403,179, 12/106,876, 11/827,637, 10/039,393, 10/254,126, 09/867,924,12/570,977, and 12/645,081; U.S. Provisional Patent Application Ser.Nos. 61/140,480, 61/091,608, 61/097,780, 61/101,461, 61/251,244,60/604,321, 60/690,652, 60/707,991, 60/208,728, 60/718,448, 61/035,949,60/830,237, and 60/925,181; and International Patent Application Nos.PCT/US2010/042556, PCT/US2001/015204, PCT/US2005/030278,PCT/US2009/069257, PCT/US2009/036992, PCT/US2009/059071,PCT/US2007/015976, and PCT/US2008/061023, each herein incorporated byreference in their entireties.

Example 2 Clickable Multifunctional Small Molecules for SurfaceFunctionalization of PAMAM Dendrimer

General Information

¹H NMR spectra were obtained using a Varian Inova 500 MHz spectrometer.Matrix-assisted laser desorption ionization time-of-flight mass spectra(MALDI-TOF-MS) were recorded on a PE Biosystems Voyager System 6050,using α-cyano-4-hydroxycinnamic acid as the matrix; electrosprayionization mass spectra (ESI-MS) was recorded using a Micromass QuattroII Electronic HPLC/MS/MS mass spectrometer. HPLC analyses were performedon a Varian Prepstar system for analysis and preparation (VarianInstruments Inc., USA) using a reverse phase HPLC column (Waters,Atlantis C18).

Ultra Performance Liquid Chromatography

HPLC analysis was carried out on a Waters Acquity Peptide Mapping Systemequipped with a Waters photodiode array detector, a column manager thatfacilitates 4 column housing, and a sample manager. The instrument iscontrolled by Empower 2 software. For characterization, calibration andquantitation 45 studies, G5 PAMAM dendrimer, its conjugates, free folicacid (FA) and methotrexate (MTX) were ran on an Acquity BEH C4 column(100×2.1 mm, 1.7 μm). The analysis was carried out using a gradientelution beginning with 99:1 (v/v) water/acetonitrile (ACN) reaching20:80 water/ACN in 13.40 minutes. The 50 gradient was thenreequilibrated back to starting conditions in the next 1.0 minute. Flowrate was maintained at 0.208 mL/min and Trifluoroacetic acid (TFA) at0.14 wt % concentration was added in water as well as in ACN as acounter ion. A 3 μL of sample was injected using a “partial loop withneedle overfill”, an inbuilt 55 sample loop option within the software.The column temperature was maintained at 35 C. The concentration of G5PAMAM dendrimer and dendrimer-ligand conjugate were maintained at 1mg/mL.

Semi_Prep Reverse Phase High Performance Liquid Chromatography

HPLC isolation was carried out on a Waters Delta 600 HPLC systemequipped with a Waters 2996 photodiode array detector, a Waters 2707auto sampler, and Waters Fraction collector III. The instrument wascontrolled by Empower 2 software. For purification of the conjugates, anAtlantis Prep T3 column (250×10 mm, 5 μl) was used. The mobile phase forelution of the conjugates was a modified two step linear gradientbeginning with 90:10 (v/v) water/ACN and ending with 75:25 (v/v)water/ACN over 20 min at a flow rate of 4.02 mL/min. In the next 10minutes, gradient was gradually changed to 25:75 (v/v)water/acetonitrile. The system was then reequilibrated back to thestarting conditions. TFA at 0.14 wt % concentration in water as well asin ACN was used as a counter ion. The HPLC system is connected to aWaters Fraction Collector III. The fractions of the intended peaks werecollected using a time program. The delay time was calculated to be 4seconds.

Stability Test

15 (100 μM) was incubated with RPMI medium containing final 10% serum.The solution was stirred continuously and maintained at 37° C. fordifferent time periods and the medium proteins were precipitated with10% DMSO in acetonitrile. The supernatant obtained were subjected HPLCanalysis UV absorbance peak area of MTX and FA was used to derive acalibration and used to determine the concentration of released FA andMTX.

Simulation Method

MD simulations were carried out on a G5 PAMAM dendrimer conjugate 15 atneutral solution to investigate structural behaviors of the dendrimer. Amolecular structure of G5 PAMAM dendrimer was generated by a recursivescript in CHARMM. 73% of the dendrimer surface was acetylated and therest of the amino groups were protonated. All hydrogens were explicitlyincluded. The MD simulations were carried out using the molecularsimulation program CHARMM version 35 (see, e.g., Brooks, B. R.; et al.,J. Comp. Chem. 1983, 4, 187-217; herein incorporated by reference in itsentirety) and the CHARMM 27 all-atom topology and parameters (see, e.g.,MacKerell, A. D.; et al., J. Phys. Chem. B 1998, 102, 3386-3616; hereinincorporated by reference in its entirety). The simulations wereperformed in distance-dependent dielectric constant (D=r) (see, e.g.,Pickersgill, R. W. Protein Eng. 1988, 2, 247-248; herein incorporated byreference in its entirety) without non-bonded cutoff distance (r_(c)=∞),which was proved to be a good simulation condition for non-hydratedPAMAM dendrimer systems (see, e.g., Lee, I.; et al., Macromolecules2002, 35, 4510-4520; herein incorporated by reference in its entirety).The total potential energy function used in the CHARMM program for MDcalculations is described as

U = U_(bonded) + U_(nonbonded)$U_{nonbonded} = {\sum\limits_{nonbonded}\{ {{ɛ\lbrack {( \frac{\sigma}{r_{ij}} )^{12} - ( \frac{\sigma}{r_{ij}} )^{6}} \rbrack} + \frac{q_{i}q_{j}}{D \cdot r_{ij}}} \}}$where ∈ is the minimum energy of Lennard-Jones potential, σ is thedistance to give minimum Lennard-Jones potential, q is the partialcharge on the atom, D is the dielectric constant, i, j are non-bondedatom pairs and r is the distance between i and j.

After the initial molecular structure was minimized by steepest descentalgorithm for 50000 steps and adopted basis Newton-Raphson algorithm for50000 steps, the system was equilibrated at 300 K for 200 ps, followedby dynamics run for 200 ps. The Verlet algorithm was used with a 1-fstime step in the MD simulations.

Materials

All solvents and chemicals were of reagent grade quality, purchased fromSigma-Aldrich Chemical Co. and used without further purification unlessotherwise noted. Thin-layer Chromatography (TLC) and columnchromatography were performed with 25 DC-Plastikfolien Kieselgel 60 F254(Merck), and Baxter silica gel 60 Å (230-400 mesh), respectively.

Synthesis

1-azido-3-aminopropane (see, e.g., Tomalia, et al., Adv Matter 1994 6529; herein incorporated by reference in its entirety),3-azido-7-hydroxy-2H-chromen-2-one (see, e.g., Sivakumar et al. (2004)Organic Lett. 6:4603-4606; herein incorporated by reference in itsentirety), and tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate wereprepared as reported (see, e.g., Sivakumar et al. (2004) Organic Lett.6:4603-4606; Carboni et al. (1993) 58:3736-3741; each hereinincorporated by reference in its entirety).

N-(3-azidopropyl)-4,6-dichloro-1,3,5-triazin-2-amine (1)

To a solution of cyanuric chloride (18.42 g, 0.10 mol) in acetone (180mL) in an ice-water bath was added diisopropylethylamine (DIPEA) (12.92g, 0.10 mol). 1-azido-3-aminopropane (5.00 g, 0.050 mol) in acetone (50mL) was added slowly over 2 hours. The reaction was stirred at roomtemperature overnight. The solvent was removed under reduced pressure.The residue was dissolved in CH₂Cl₂, washed with water, dried (Na₂SO₄)and rotary evaporated. The resulting residue was purified by columnchromatography on silica gel (eluent CH₂Cl₂) to give 1 as a white solid(5.62 g, 45%): ESI-MS m/z 248.1 (M+H⁺) calculated for C₆H₈Cl₂N₇ 248.0.

2-(4-(3-azidopropylamino)-6-chloro-1,3,5-triazin-2-ylamino)ethanol (2)

Ethanolamine (2.47 g, 40.4 mmol) in acetone (20 mL) was added to asolution of 1 (2.0 g, 8.1 mmol) in acetone (30 mL). The reaction wasstirred at room temperature for 24 hours. The acetone was removed byrotary evaporation and the residue was suspended in CH₂Cl₂. The mixturewas filtered off. The solid collected was washed with CH₂Cl₂ with H₂Oand dried under reduced pressure to afford 2 as a white solid (1.86 g,85%). The product is sufficiently pure for further reactions. ESI-MS m/z273.1 (M+H⁺) calculated for C₈H₁₄ClN₈O 273.1.

Tert-butyl2-(2-(4-(3-azidopropylamino)-6-(2-hydroxyethylamino)-1,3,5-triazin-2-ylamino)ethoxy)ethylcarbamate(3)

DIPEA (142 mg, 1.10 mmol), tert-butyl2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (273 mg, 1.10 mmol), and 2(150 mg, 0.55 mmol) were dissolved in THF (5 mL). The mixture wasstirred at 70° C. under N₂ overnight. The solvent was removed underreduced pressure. The resulting residue was purified by columnchromatography on silica gel (eluent CH₃OH/CH₂Cl₂=2/98) to give 3 as awhite solid (124 mg, 47%): ESI-MS m/z 485.2 (M+H⁺) calculated forC₁₉H₃₇N₁₀O₅ 485.3.

2-(4-(2-(2-aminoethoxy)ethylamino)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethanol(4)

(485 mg, 1.0 mmol) was dissolved in CH₂Cl₂ (10 mL). TFA (1 mL) was addedand the mixture was stirred at room temperature overnight. The solventwas removed under reduced pressure. And the residue was dissolved in H₂O(20 mL) and filtered off. 1N NaOH was added dropwise to the filtrateuntil the solution became basic. CH₂Cl₂ (20 mL) was added and theorganic layer was washed with brine and H₂O, dried over Na₂SO₄, filteredand evaporated under reduced pressure to yield 4 as a white solid (358mg, 93%): ESI-MS m/z 385.2 (M+H⁺) calculated for C₁₄H₂₉N₁₀O₃ 385.2.

2-(4-(2-(2-aminoethoxy)ethylFA)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethanol(5)2-(4-(2-(2-aminoethoxy)ethylFA)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethylMTX(6)

Folic acid (FA) (195 mg, 0.44 mmol) in DMSO (10 mL) was added to asolution of 4 (170 mg, 0.44 mmol) and (DCC) (182 mg, 0.88 mmol) in DMSO(5 mL). The reaction was stirred at room temperature for 24 hours. Thereaction mixture was filtered. H₂O (30 mL) was added to the filtrate.The resulting precipitate was filtered, washed with H₂O and acetone, anddried under reduced pressure. The cruder product was further purified byHPLC. Compound 5 was obtained as a yellow solid (147 mg, 41%): ESI-MSm/z 808.3 (M+H⁺) calculated for C₃₃H₄₆N₁₇O₈ 808.4.

Methotrexate (MTX) (25 mg, 0.055 mmol) in DMSO (5 mL) was added to asolution of 5 (40 mg, 0.50 mmol), N,N′-dicyclohexylcarbodiimide (DCC)(204 mg, 1.0 mmol), and trace amount 4-(dimethylamino)pyridine (DMAP) inDMSO (5 mL). The reaction was stirred at room temperature for 24 hours.H₂O (30 mL) was added to the reaction mixture. The solid was collectedby centrifugation, washed with H₂O and acetone, and dried under vacuumto yield 6 as a yellow solid.

Two isomers 5 and 6 were isolated and purified as yellow solids, 5 (94mg 26%), 6 (53 mg, 15%): ¹H NMR (500 MHz, d₆-DMSO) 5 δ 1.23 (m, 1H),1.77 (m, 2H), 1.85 (m, 1H), 1.96 (m, 1H), 2.26 (m, 2H), 3.49 (m, 25H),4.35 (m, 1H), 4.50 (s, 1H), 6.63 (d, J=8.5 Hz, 2H), 7.28 (m, 2H), 7.65(d, J=8.5 Hz, 2H), 7.87-7.98 (m, 2H), 8.10 (m, 1H), 8.27 (m, 1H), 8.66(s, 1H); 6 δ 1.23 (m, 1H), 1.76 (m, 2H), 1.85 (m, 1H), 2.04 (m, 1H),2.19 (m, 2H), 3.17 (t, J=6.0 Hz, 1H), 3.35-3.61 (m, 24H), 4.28 (m, 1H),4.50 (s, 1H), 6.64 (d, J=8.5 Hz, 2H), 7.22 (m, 2H), 7.65 (d, J=8.5 Hz,2H), 7.89 (m, 1H), 8.11 (m, 1H), 8.19 (d, J=7.5 Hz, 1H), 8.27 (m, 1H),8.66 (s, 1H); ESI-MS m/z 808.3 (M+H⁺) calculated for C₃₃H₄₆N₁₇O₈ 808.4.

(R)-2-(4-((2-amino-4-oxo-1,4-dihydropteridin-6-yl)methylamino)benzamido)-5-(2-(2-(2-aminoethoxy)ethoxy)ethylamino)-5-oxopentanoicacid (7) (see FIG. 5)

Folic acid (882 mg, 2.0 mmol) in DMSO (30 mL) was added to a solution oftert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (496 mg, 2.0 mmol)and DCC (620 mg, 3.0 mmol) in DMSO (10 mL). The reaction was stirred atroom temperature for 24 hours. The reaction mixture was filtered. H₂O(50 mL) was added to the filtrate. The resulting precipitate wasfiltered, washed with H₂O and acetone, and dried under reduced pressure.The solid collected was suspended in H₂O (30 mL) and TFA (15 mL) wasadded inside. The mixture was stirred at room temperature overnight, andthen filtered. The filtrate was evaporated to dryness under reducedpressure. Compound 7 was obtained as a yellow solid (874 mg, 62%):ESI-MS m/z 572.0 (M+H⁺) calculated for C₂₅H₃₄N₉O₈ 572.3.

3-azido-7-(4,6-dichloro-1,3,5-triazin-2-yloxy)-2H-chromen-2-one (8) (seeFIG. 5)

To a solution of cyanuric chloride (2.27 g, 12.3 mmol) in acetone (30mL) in an ice-water bath was added diisopropylethylamine (DIPEA) (1.59g, 12.3 mmol). 3-azido-7-hydroxy-2H-chromen-2-one (1.00 g, 4.9 mmol) inacetone (30 mL) was added slowly over 2 hours. The reaction was stirredat room temperature overnight. The solvent was removed under reducedpressure. The residue was dissolved in CH₂Cl₂, washed with water, dried(Na₂SO₄) and rotary evaporated. The resulting residue was purified bycolumn chromatography on silica gel (eluent CH₂Cl₂) to give 8 as a paleyellow solid (1.02 g, 59%): ESI-MS m/z 351.0 (M+H⁺) calculated forC₁₂H₅Cl₂N₆O₃ 351.0.

3-azido-7-(4-chloro-6-(2-hydroxyethylamino)-1,3,5-triazin-2-yloxy)-2H-chromen-2-one(9) (see FIG. 6)

Ethanolamine (45 mg, 0.74 mmol) in acetone (20 mL) was added to asolution of 8 (173 mg, 0.49 mmol) and DIPEA (96 mg, 0.74 mmol) inacetone (10 mL). The reaction was stirred at room temperature for 24hours. The acetone was removed by rotary evaporation and the residuepurified by column chromatography on silica gel (eluentCH₃OH/CH₂Cl₂=1/99) to give 9 as a pale yellow solid (144 mg, 78%):ESI-MS m/z 376.1 (M+H⁺) calculated for C₁₄H₁₁ClN₇O₄ 376.1.

7-(4-(2-(2-(2-aminoethoxy)ethoxy)ethylFA)-6-(2-hydroxyethylamino)-1,3,5-triazin-2-yloxy)-3-azido-2H-chromen-2-one(10) (see FIG. 6)

DIPEA (9 mg, 0.07 mmol), 7 (13 mg, 0.02 mmol), and 9 (9 mg, 0.02 mmol)were dissolved in DMSO (2 mL). The mixture was stirred at 60° C. underN₂ overnight. The reaction mixture was filtered. H₂O (10 mL) was addedto the filtrate. The resulting precipitate was filtered, washed with H₂Oand acetone, and dried under reduced pressure. The cruder product wasfurther purified by HPLC. Compound 10 was obtained as a yellow solid:ESI-MS m/z 909.2 (M−H⁺) calculated for C₃₉H₄₁N₁₆O₁₁ 909.3.

Synthesis of (R)-5-(2-(tert-butoxycarbonylamino)ethyl) 1-tert-butyl2-(4-(((2,4-diaminopteridin-6-yl)methyl)(methyl)amino)benzamido)pentanedioate(MTX-α-OtBu-γ-C₂NHBoc) (10) (see FIG. 6)

MTX-α-OtBu 9 (80 mg, 0.16 mmol) in DMF (2 mL) was added to a solution oftert-butyl 2-hydroxyethylcarbamate (38 mg, 0.24 mmol),2-chloro-1-methyl-pyridinium iodide (CMPI) (44 mg, 0.17 mmol), and4-(dimethylamino)pyridine (DMAP) (44 mg, 0.36 mmol) in DMF (3 mL). Thereaction was stirred at room temperature for 6 hours. The solvent wasremoved under reduced pressure. The residue was dissolved in CH₂Cl₂,washed with water, dried (Na₂SO₄) and rotary evaporated. The resultingresidue was purified by column chromatography on silica gel (eluentCH₃OH/CH₂Cl₂=6/94) to give 10 as a yellow solid (30 mg, 30%): ¹H NMR(500 MHz, d₄-THF) δ 1.38 (s, 9H), 1.45 (s, 9H), 1.88 (m, 1H), 2.21 (m,1H), 2.39 (m, 2H), 2.54 (s, 2H), 3.19 (s, 3H), 3.25 (m, 2H), 3.93 (m,1H), 4.12 (m, 1H), 4.66 (m, 1H), 4.75 (s, 2H), 6.43 (t, J=5.5 Hz, 1H),6.79 (d, J=8.5 Hz, 2H), 7.08 (s, 2H), 7.38 (d, J=8.0 Hz, 1H), 7.74 (d,J=9.0 Hz, 2H), 8.53 (s, 1H), 10.9 (s, 1H); ESI-MS m/z 654.3 (M+H⁺)calculated for C₃₁H₄₄N₉O₇ 654.3.

Synthesis of(R)-5-(2-aminoethoxy)-2-(4-(((2,4-diaminopteridin-6-yl)methyl)(methyl)amino)benzamido)-5-oxopentanoicacid (MTX-α-OtBu-γ-C₂H₂) (11) (see FIG. 6)

10 (30 mg, 0.046 mmol) was dissolved in 4 mL CH₂Cl₂/TFA (1:1) and themixture was stirred at room temperature for 1 hour. The solvent wasremoved under reduced pressure. Ether (10 mL) was added to the residue.The solid was collected by centrifugation, washed with H₂O and acetone,and dried under vacuum to 3 as a yellow solid (21 mg, 92%). The productis sufficiently pure for further reactions: ESI-MS m/z 496.3 (M−H⁺)calculated for C₂₂H₂₆N₉O₅ 496.2.

Synthesis of2-(4-(2-(2-aminoethoxy)ethylFA)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethyl(4-nitrophenyl) carbonate (Triazine-N₃—OC₆H₄NO₂-αFA) (7) (see FIG. 5)

5 (20 mg, 0.025 mmol) was dissolved in DMSO (1 mL), Triethylamine (10μl) was added to the mixture, followed by bis(4-nitrophenyl)carbonate(15 mg, 0.049 mmol). The mixture was kept at room temperature for 48 h.Ether (10 mL) was added to the residue. The solid was collected bycentrifugation, washed with CH₂Cl₂, methanol, and acetone, and driedunder vacuum to give 7 as a yellow solid. The sample was used for thenext step without further purification.

Synthesis of2-(4-(2-(2-aminoethoxy)ethylFA)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethylMTX(Triazine-N₃-γMTX-αFA) (8) (see FIG. 5)

11 (25 mg, 0.050 mmol) in DMSO (0.5 mL) was added to a solution of 7 andDIPEA (32 mg, 0.25 mmol) in DMSO (0.5 mL). The reaction was stirred atroom temperature for 24 hours. Ether (10 mL) was added to the residue.The solid was collected by centrifugation, washed with methanol andacetone, and dried under vacuum. The cruder product was further purifiedby semi-prep. HPLC to yield 8 as yellow solids (12 mg, 36%): ¹H NMR (500MHz, d₆-DMSO) δ 1.74 (m, 2H), 1.83 (m, 1H), 1.95 (m, 2H), 2.08 (m, 2H),2.17 (m, 2H), 2.39 (m, 3H), 3.07-3.46 (m, 28H), 3.95-4.03 (m, 2H),4.18-4.25 (m, 2H), 4.31 (m, 2H), 4.48 (m, 2H), 4.78 (m, 2H), 6.62 (m,4H), 6.82 (m, 3H), 6.95 (m, 1H), 7.22 (m, 1H), 7.46 (m, 2H), 7.62-7.73(m, 5H), 7.86 (m, 1H), 7.97 (m, 1H), 8.56 (s, 1H), 8.63 (s, 1H); ESI-MSm/z 664.3 (M−2H⁺)/2 calculated for (C₅₆H₇₀N₂₆O₁₄-2H⁺)/2 664.3.

Synthesis of tert-butyl(2-(2-(2-((4-((3-azidopropyl)amino)-6-((2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)amino)-1,3,5-triazin-2-yl)amino)ethoxy)ethoxy)ethyl)carbamate(Triazine-N₃—OC₆H₄NO₂—NHBoc) (12) (see FIG. 7)

3 (158 mg, 0.33 mmol) was dissolved in DMF (3 mL). Triethylamine (200μA) was added to the mixture, followed by bis(4-nitrophenyl)carbonate(198 mg, 0.65 mmol). The mixture was kept at room temperature for 24 h.The solvent was removed under reduced pressure. The resulting residuewas purified by column chromatography on silica gel (eluentCH₃OH/CH₂Cl₂=5/95) to give 12 as a white solid (182 mg, 86%): ¹H NMR(500 MHz, CDCl₃) δ 1.45 (s, 9H), 1.84 (m, 2H), 2.17 (s, 1H), 3.32-3.77(m, 18H), 4.42 (m, 2H), 5.25 (m, 1H), 5.94 (m, 1H), 6.30-6.48 (m, 1H),7.37 (d, J=9.0 Hz, 2H), 8.27 (d, J=8.5 Hz, 2H); ESI-MS m/z 650.2 (M+H⁺)calculated for C₂₆H₄₀N₁₁O₉ 650.3.

Synthesis of2-(4-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate)-6-(3-azidopropylamino)-1,3,5-triazin-2-ylamino)ethylMTX(Triazine-N₃-MTX-NHBoc) (13) (see FIG. 7)

11 (10 mg, 0.020 mmol) in DMF (0.5 mL) was added to a solution of 12 (26mg, 0.040 mmol) and DIPEA (26 mg, 0.20 mmol) in DMF (0.5 mL). Thereaction was stirred at room temperature for 24 hours. CH₂Cl₂ (5 mL) wasadded to the residue. The solid was collected by centrifugation, washedwith acetone and CH₂Cl₂, and dried under vacuum to give 13 as a yellowsolid (7 mg, 35%). The sample was used for the next step without furtherpurification: ESI-MS m/z 1006.5 (M+H⁺) calculated for(C₅₆H₇₀N₂₆O₁₄C₃₃H₄₆N₁₇O₈+H⁺) 1006.5.

General Procedure for Synthesis of Dendrimer Conjugates Synthesis ofG5-NHAc₈₀-Alkyne₁₂ (14) (see FIG. 8)

Compound was synthesized according to the literature.⁷G5-NHAc₈₀-Alkyne₁₂ 14 White solid: MALDI-TOF mass 32774. The ¹H NMRintegration determined the mean number of Acetyl groups per dendrimer is80.1. The mean number of Alkyne ligands per dendrimer is 12.3.

¹H NMR Analyses to Determine the Degree of Functionalization

The number of ligands that attached to the dendrimer were determined bycomparing the integration of the methyl protons of the terminal acetylgroups to the aromatic protons on the conjugated ligands (see, e.g.,Mullen, D. G.; Acs Nano 2010, 4, 657-670; herein incorporated byreference in its entirety). The number of acetyl groups per dendrimerwas independently determined by first computing the total number of endgroups from the number average molecular weight (GPC) and potentiometrictitration data for G5-NH₂(100%) as previously described (see, e.g.,Majoros, I. J.; et al., J Med Chem 2005, 48, 5892-5899; hereinincorporated by reference in its entirety). The total number of endgroups was applied to the ratio of primary amines to acetyl groups,obtained from the ¹H NMR of the partially acetylated dendrimer, tocompute the average number of acetyl groups per dendrimer.

General Procedure for Cu(I) Catalyzed Cycloaddition

G5-NHAc₈₀-Alkyne₁₂ 14 (see FIG. 8) (14 mg, 0.43 μmol) was dissolved inCu(II) sulfate (10 mol % per triazine-azide, 1 mg/mL H₂O) and sodiumascorbate (60 mol % per triazine-azide, 1 mg/mL H₂O solution) solution.The Triazine-azide substrates (5, 8, and 13), (7.5 azide mole ratio toG5-NHAc₈₀-Alkyne₁₂, 10 mg/mL DMSO solution) were added. The reactionmixture was heated in an oil bath at 50° C. overnight. Samples werepurified using 10 000 MWCO centrifugal filtration devices. Purificationconsisted of ten cycles (20 min at 4800 rpm) using PBS (5 cycles) and DIwater (5 cycles). The purified dendrimer samples were lyophilized toyield 15, 16, and 17 (FIG. 8) as yellow solids.

G5-Triazine-γMTX_(3.1)-αFA_(3.1) 15 (FIG. 8): MALDI-TOF mass 35094. The¹H NMR integration determined the mean number of Triazine-γMTX-αFAattached is 3.1.

G5-Triazine-OH-αFA_(4.2) 16 (FIG. 8): MALDI-TOF mass 33059. The ¹H NMRintegration determined the mean number of Triazine-OH-αFA is 4.2.G5-Triazine-γMTX_(3.7)-NHBoc 17 (FIG. 8): MALDI-TOF mass 34266. The ¹HNMR integration determined the mean number of Triazine-γMTX-NHBoc is3.7.

Example III

This example describes the synthesis of triazine derivatives, theirconjugation to PAMAM dendrimers, and in vitro cytotoxicity studies onFR-expressing KB cells using these dendrimer conjugates.

Trifunctional triazine derivatives were synthesized from cyanuricchloride by consecutive aromatic nucleophilic substitution reactions,which were controlled by temperature to run in a stepwise manner (FIG.5). Folic acid (FA) was conjugated to the triazine through an amidebond. An ester bond was chosen for methodtrexate (MTX) conjugation withexpectation for improved release of drug. The monosubstituted triazineTriazine-N₃ 1 was synthesized by adding 3-azidopropan-1-amine to asolution of cyanuric chloride in acetone at 0° C. The secondsubstitution was carried out in the same solvent at room temperaturewith 2-aminoethanol. The bisubstituted Triazine-N₃—OH 2 was treated withtert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate in the presenceof diisopropylethylamine (DIPEA) in tetrahydrofuran (THF) at 70° C. toform Trazine-N₃—OH—NHBoc 3 containing a primary amine in a protectedform, which was used for conjugation with FA. Reaction of 2 withunprotected 2,2′-(ethane-1,2-diylbis(oxy))diethanamine had a poor yielddue to a major by-product of the triazine dimer. After removal of theBOC protecting group, FA was coupled to Triazine-N₃—OH—NH₂ 4 byN,N′-Dicyclohexylcarbodiimide (DCC) in dimethylsulfoxide (DMSO). Thissample was purified on RP-HPLC. Two isomers, 5 and γ-carboxyl 6 grouplinked products were separated and purified, and relatively higher (60%)α-carboxyl linked product was obtained vs. γ isomer (40%), based on HPLCanalysis. Both isomers were conjugated to PAMAM dendrimers. Resultingconjugates showed similar binding affinities to purified FR-α in asurface plasmon resonance (SPR)-based binding study. This finding wasconsistent with other research group's results (see, e.g., Leamon, C.P.; et al., J Drug Target 1999, 7, 157; Bettio, A.; et al., eachincorporated by reference in their entireties). The HPLC-purifiedα-carboxyl group linked isomer 5 was used for further studies due to itsrelatively higher yield.

The strategy for the synthesis of trifunctional Triazine-N₃-γMTX-αFA 8was first approached by direct coupling MTX with Triazine-N₃—OH-αFA 5using DCC/4-(dimethylamino)pyridine (DMAP) or DCC/4-pyrrolidinopyridine(see, e.g., Devineni, D.; et al., Bioconjugate Chem 1995, 6, 203; hereinincorporated by reference in its entirety). However, both reactions wereunsuccessful due to very low yield. To solve this problem, an alternatesynthetic route was chosen (FIG. 5). The hydroxyl group of 5 was firstactivated with bis(4-nitrophenyl)carbonate to provide carbonate 7. Thencarbonate 7 was treated with γMTX-ethylamine 11 (FIG. 6) to form thetarget compound Triazine-N₃-γMTX-αFA 8. Control sampleTriazine-N₃-γMTX-NHBoc 13 was synthesized in a similar manner (FIG. 7).

γMTX-ethylamine 11 was synthesized following the literature (see, e.g.,Nagy, A.; et al., P Natl Acad Sci USA 1993, 90, 6373; hereinincorporated by reference in its entirety). In brief,(benzotriazol-1-yloxytris(dimethylamino) phosphoniumhexafluorophosphate) (BOP) activated 4-amino-4-deoxy-N¹⁰-methyl pteroicacid (APA) in DMSO was reacted with the potassium salt of glutamic acidα-tert-butyl ester to form the α-carboxyl protected MTX derivative. Theselectively protected MTX derivative was then coupled to tert-butyl(2-hydroxyethyl)carbamate by 2-chloro-1-methyl-pyridinium iodide(CMPI)/DMAP. The t-butyl and BOC protecting groups were then removed bytrifluoroacetic acid (TFA) at the same time to afford theγMTX-ethylamine 11. Alkyne-terminated generation 5 (G5) PAMAM dendrimerG5-NHAc-Alkyne 14 has been previously synthesized (see, e.g., Mullen, D.G.; et al., Acs Nano 2010, 4, 657; herein incorporated by reference inits entirety). Partially acetylated dendrimers was employed in thisstudy because these materials have been shown to be most effective fortargeted drug delivery applications (see, e.g., Thomas, T. P.; et al., JMed Chem 2005, 48, 3729;

Kukowska-Latallo, J. F.; et al., Cancer Res 2005, 65, 5317; each hereinincorporated by reference in their entireties). The parentamine-terminated materials have been shown to non-selectively cause cellmembrane permeability and are not suited for in vivo targeted drugdelivery applications.³¹⁻³³ Target conjugate G5-Triazine-γMTX-αFA 15,control samples G5-Triazine-OH-αFA 16, and G5-Triazine-γMTX-NHBoc 17were synthesized by one step “click” reaction to the G5-Ac-Alkyne 14with corresponding triazine derivatives (FIG. 8). MALDI-TOF and NMRanalyses were performed to determine the degree of functionalization.

The cytotoxicity of the newly synthesized G5-Triazine-γMTX-αFA 15 wasdetermined in vitro by the XTT assay in KB cells, which express a highcell surface FR (FIG. 9). The cytotoxicity with another batch ofG5-FA-MTX synthesized by Cambrex Inc. was also compared, using theclassic sequential conjugation synthetic pathway (see, e.g., Majoros, I.J.; et al., J Med Chem 2005, 48, 5892; herein incorporated by referencein its entirety). Previous studies have shown that this batch of Cambrexconjugates was cytotoxic in vitro and tumoricidal in vivo, with potencysimilar to that of our previously published compound (see, e.g., Thomas,T. P.; et al., J Med Chem 2005, 48, 3729; Kukowska-Latallo, J. F.; etal., Cancer Res 2005, 65, 5317; each herein incorporated by reference intheir entireties). The newly synthesized G5-Triazine-γMTX-αFA 15inhibited the KB cell growth in a dose-dependence fashion, withincreased cytotoxic potential vs. the Cambrex batch. TheG5-Triazine-γMTX-αFA 15 and the Cambrex conjugate inhibited cells growthwith IC50 of ˜5 and 20 nM, respectively. The Cambrex conjugate has 4-5FA and 7-8 MTX per dendrimer, while 15 has 3.1 each of the FA and MTXper dendrimer. Despite the increased MTX content on the Cambrexconjugate, 15 carried higher cytotoxic potential. As MTX has ˜1000× foldmore affinity toward dihyrofolate reductase (DHFR) vs. FA, it ispossible that the close molecular proximity of the FA and MTX on 15, butnot on the Cambrex conjugate, may allow the preferential binding of MTXon 15 as it is being juxtaposed at the DHFR binding site. The controlconjugate G5-Triazine-γMTX 17 without the targeting moiety FA alsoshowed a dose dependent cytotoxicity, though with a much higher IC50(˜75 nM) vs. G5-Triazine-γMTX-αFA 15. As the G5-Triazine-γMTX conjugateis unlikely to be taken up through the reduced folate carrier (RFC) dueto size constraints of this membrane channel, it is possible that it isinternalized into the cells through polyvalent binding through the FR.

In order to confirm that the observed cytotoxicity was not due to freeMTX released from the conjugate during incubation in the cell culturemedium, the stability of the dendrimer conjugate was tested in the cellculture medium. For this analysis, the G5-Triazine-γMTX-αFA 15 wasincubated with cell culture medium for 1, 4, 24, and 72 h. During the 72h incubation time periods, both FA and MTX showed a slow release in atime-dependent fashion (FIG. 10). Amide-linked FA was quite stableagainst hydrolytic cleavage, whereas the ester-linked MTX was moreliable to hydrolysis, 1.0% FA vs. 4.6% MTX, for the 72 h period studied.This data shows that the major antiproliferative effect of theG5-Triazine-γMTX-αFA 15 was not the result of free MTX.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled indendrimer synthesis, drug delivery, or related fields are intended to bewithin the scope of the following claims.

We claim:
 1. A composition comprising a PAMAM dendrimer conjugated witha triazine compound, wherein said triazine compound is selected from thegroup consisting of


2. The composition of claim 1, wherein said PAMAM dendrimer isconjugated with said triazine compound via a 3-azido-coumarine linkageagent.
 3. The composition of claim 1, wherein said triazine compound isconjugated with one or more ligands.
 4. The composition of claim 3,wherein said one or more ligands are independently selected from thegroup consisting of a therapeutic agent, a targeting agent, an imagingagent, and a trigger agent.
 5. The composition of claim 3, wherein saidone or more ligands are methotrexate, folic acid, and 3-azido-coumarine.6. A method of synthesizing a functionalized dendrimer, said methodcomprising: a) providing an alkyne-modified PAMAM dendrimer and atriazine compound, wherein said triazine compound is selected from thegroup consisting of

and b) conjugating said alkyne-modified PAMAM dendrimer with saidtriazine compound.
 7. The method of claim 6, wherein conjugating of saidalkyne-modified PAMAM dendrimer with said triazine compound occurs via aclick chemistry reaction.
 8. The method of claim 6, wherein said PAMAMdendrimer is conjugated with said triazine compound via a3-azido-coumarine linkage agent.
 9. The method of claim 6, furthercomprising step c) conjugating one or more ligands with said triazinecompound.
 10. The method of claim 9, wherein said one or more ligandsare selected from the group consisting of a therapeutic agent, atargeting agent, an imaging agent, and a trigger agent.
 11. The methodof claim 9, wherein said one or more ligands are methotrexate and folicacid.